Traditional Medicinal Plants (Dar Es Salaam University Press - Ministry of Health - Tanzania, 1991, 391 p.) PART I: USE AND PROMOTION OF TRADITIONAL MEDICINAL PLANTS IN THE AFRICAN REGION Registration and utilization of herbal remedies in some countries of Eastearn, Central and Southern Africa A report on the development of a traditional medicine for bronchial asthma Resume of current research in medicinal plants in Botswana The use of data from traditional medicine: Tunisian experience Chemical and pharmacological studies of marketed traditional drugs Research into medicinal plants: The Somali experience Effect of nitrogen and phosphorus on the essential oil yield and quality of chamomile (Matricaria chamomilla L.) flowers Chemical characterization of pharmacologically active compounds from Synadenium pereskiifolium Abietane diterpene quinones from lepechinia bullata Antimicrobial activity of Tanzanian traditional medicinal plants Identification of clovanediol: A rare sesquiterpene from the stem bark of canella winterana L. (Canellaceae), using spectrophotometric methods A comparative study of the traditional remedy ''Suma-kala'' and chloroquine as treatment for malaria in the rural areas Ethnobotany and conservation of medicinal plants Biotransformation of hydroxyanthraquinone glycosides in Cassia species Le médicament indigène Africaine: Sa standardisation et son évaluation dans le cadre de la politique des soins de santé primaires Chemical Evaluation of Tanzanian medicinal plants for the active constituents as a basis for the medicinal usefulness of the plants Ethnobotany and the medicinal plants of the Korup rainforest project area, Cameroon Seaweeds in medicine and pharmacy: A global perspective Biotechnology and medicinal plants Phytochemical investigations of four medicinal plants of Malawi: What next? The chemistry and pharmacology of the essential oil from the leaves of Hyptis suaveolens (L) Point Some CNS effects of Datura stramonium L (Solanaceae) in mice Discovery and development of drugs from natural sources A Survey of medicinal plants in Tabora region, Tanzania Intérêt pharmacognosique des plantes de la flore médicinale Rwandaise: valeur chimiotherapeutique de quelques plantes Rwandaise A note on the utilization and commercialisation of traditional medicine Experience on the use of Tanzanian medicinal plants for the last decade (1979-1989) A comparison of the status of medicinal plants development in Africa with selected parts of the world Expérience du Burkina Faso en matière de pharmacopeé traditionnelle The role and use of ethnomedical data in the research on traditional medicines and medicinal plants Traditional medicinal plants: Our cultural heritage The use of traditional medicinal plants: The cultural context

Traditional Medicinal Plants (Dar Es Salaam University Press - Ministry of Health - Tanzania, 1991, 391 p.)

PART I: USE AND PROMOTION OF TRADITIONAL MEDICINAL PLANTS IN THE AFRICAN REGION

Registration and utilization of herbal remedies in some countries of Eastearn, Central and Southern Africa

OLAWAYO AKERELE,
Programme Manager, Traditional Medicine
World Health Organization, Geneva

Introduction

Traditional medicine has been practised for the last several thousand years, although it found a place in the WHO programme only twelve years ago.

Traditional medicine is widespread throughout the world in a variety of forms. Its practices are based on beliefs that were in existence, often for hundreds of years, before the development and spread of modern scientific medicine, and that are still prevalent to day.

The recent development and resurgence of traditional medicine activities in the African Region grew out of the political events of the 1960s. With the advent of political independence, Africans felt the need to rediscover their sociocultural identity, and traditional medicine, an integral part of their heritage, and benefited from this return to the fountain-head. In reality, the masses had never stopped making use of traditional medicine, despite the imposition of modern medicine by the colonial powers. Moreover, economic circumstances were making imported techniques and drugs less and less accessible, forcing the authorities to take a fresh look at the problem and study the possibility of using traditional medicine to improve the health of their populations.

In most cases, however, it was necessary to convince the political decision-makers that traditional medicine had something to offer. To this end, the World Health Assembly and the Executive Board passed a number of resolutions in support of traditional medicine globally. In addition, the Regional Committee for Africa passed a number of resolutions reflecting this political will.

Three periods which correspond to very definite political and economic development stages can be distinguished in the development of traditional medicine in the African countries. These are, first, the pre-colonial period, when traditional medicine reigned supreme. Unfortunately, there was no record of traditional practices and materia medica, even though these have contributed to the modern-day therapeutic arsenal. The examples of physostigmine from the Calabar bean and the life-saving vincristine from the African periwinkle, illustrate past and present contributions. The colonial period was marked by the introduction of modern medicine and the suppression of Africa's traditional systems of medicine. Finally, the post-colonial period is represented by a renewed cultural awareness of, and pride in, traditional medicine and its values.

Primary health care has been adopted by all WHO member states, including those on the African continent, as the appropriate strategy, for developing national health systems. This approach has become imperative for technologically less advanced countries, given their present economic crisis. However, even the primary care demands the use of therapeutic preparations, and in the face of declining foreign exchange earnings, governments are finding it increasingly difficult to make essential drugs available to their rapidly growing populations. The use of medicinal plants in traditional medicine thus Finds its natural expression, and further development in primary health care, where in many cases they bridge the gap between the availability of and the demand for essential drugs. It is, however, at this level that the transition from traditional practice to medical care can most readily be made.

In our common efforts to extend coverage of the health services to improve medical care and to control major endemic and epidemic diseases we have often not fully recognized just how important a role medicinal plants play in the health of the peoples of the world. In developing countries, about three-quarters of the population rely on medicinal plants for their primary health care. In technologically advanced societies, consumers preference is shifting from synthetic to natural products and this is dictating the pace of the resurgence and expansion of the use of medicinal plants in therapy in industrialized countries. It is only logical for WHO to collaborate with others to develop activities in this exciting area of manufacture and promotion of the use of new drugs of plant origin by encouraging countries to make fuller use of the natural wealth of medicinal plants, which most of them possess. Some of the currently known herbal medicinal products could substitute imported drugs, which currently require foreign exchange for their purchase. In addition, plants used in traditional medicine hold a great, but still largely unexplored potential, for the development of new drugs against major diseases, such as AIDS, for which no safe, effective treatment is as yet available.

As part of those efforts by WHO, a workshop was organized by the Organization's Programme for Traditional Medicine, in collaboration with the Danish International Development Agency (DANIDA) and hosted by the Ministry of health of Zimbabwe. It was held from 26 June to 6 July 1989 at Kadoma, Zimbabwe.

The workshop was attended by participants from East, Central and Southern Africa and included scientists from Botswana, Kenya, Lesotho, Malawi, Swaziland, United Republic of Tanzania, Zambia and Zimbabwe, representing a variety of disciplines that are crucial to initiating multidisciplinary research in drug development from herbal remedies. These include: pharmacy, pharmacology, phytochemistry, health administration, and clinical sciences.

The workshop was the first of a series for the African Region and was intended to address issues hindering the introduction of traditional remedies into national health systems. Key issues discussed included ensuring safety and efficacy of traditional remedies, as well as associated problems of standards, stability, and dosage formulation. Safety is, indeed, a crucial issue. It is often erroneously believed that products that are natural carry no risk to the consumer. Nothing could be further from the truth. Much of our present-day powerful therapeutic arsenal is derived from plants and plant products.

This workshop was designed to establish a logical "thought process" for decision-making that is related to the utilization of herbal preparations as drugs. The workshop began with presentations from each participating country on the use of traditional medicine. A summary of the current situation with regard to traditional practitioners and the registration of herbal remedies is given below: a series of formal lectures followed, addressing areas such as the importance of medicinal plants in therapy; development of a traditional medicine pharmacopoeia; types and sources of information available on medicinal plants and their chemical constituents; how the information can be evaluated; safety and toxicological testing procedures; and the planning of clinical studies. All participants were then provided with copies of original articles on commonly used plants and challenged to decide whether each could be introduced into their national health system. Using a well-defined decision-making process, the participants answered questions about the safety and efficacy of the plants and categorized them as meriting acceptance without further study, requiring further work, or meriting outright rejection on grounds of toxicity.

It is widely believed that the use of medicinal plants in health care is increasing in the African region, and that trade in these substances is on the rise. However, no valid data are currently available on utilization and trade patterns. Plant-derived remedies currently in use range from traditional preparations such as decoctions to locally manufactured modern formulations in the form of syrups, tablets and capsules, as well as products imported from Asia. This increase in intercontinental trade in plant- derived substances has triggered concern for regulation in countries of East, Central and southern Africa. No regulations related to the use of plant-derived remedies currently exist in these countries. However, national drug legislation to cover manufacture of herbal remedies is being contemplated in all the countries. The necessary registration process should be contingent upon review of available sources of information, quality control of raw material, modern toxicology testing, and good manufacturing practices. In addition, one of the chief contributions that traditional medicine has made and continues to make to health, is the discovery of plants of medical value. "Save Plants that Save Lives" is a call to safeguard this heritage, and regulations should therefore cover conservation measures.

Country presentations at the Workshop described the current regulatory status of traditional medicine and practitioners. This information is summarized below.

Current regulatory status in some countries of East, Central and Southern Africa

Botswana

No regulation related to the use and practice of traditional medicine exists. A provisional council has been appointed to decide what to do, and will probably propose some draft legislation regarding traditional medicine. Modern medicine must be registered in the country of origin.

Kenya

There is no regulation regarding the practice of traditional medicine. The Ministry of Culture and Social Services issues certificates to traditional practitioners, but they must also obtain the permission of the area chief to practise. There is no regulation concerning the manufacture and or use of traditional remedies.

Lesotho

National drug legislation is being formulated and will create some controls for traditional remedies. The proposed regulation will lead to the registration of traditional medicines for an initial period of 8-10 years, based on safety as the sole criterion. Subsequently, registration of traditional remedies will have to be based on efficacy as well as safety.

Malawi

The Pharmacy Medicine and Poisons Act of 1988 does not have any provision regarding the use of traditional medicinal remedies. Since traditional practitioners are not used in the health services, the need to register them has never arisen. Some other provisions of the Act are related to the exclusion of traditional practitioners from practice. For example, "no person shall sell by retail, or supply in circumstances corresponding to retail sale or administer, other than to himself, a medicinal product of a description or a class specified by Order made by the Minister and published in a Gazette except in accordance with prescription given by an appropriate practitioner," which excludes traditional practitioners.

Similarly, Section 17(1)(b) of the Act indicates that "except as is provided by this Act, no person other than a person registered as a pharmacist under this part shall in the course of any trade or business prepare, mix compounds, or dispense any medicinal product or poison except under the supervision of a registered pharmacist". Thus, it can be deduced from this provision that traditional healers should not practice their trade. In practice, however, people are not imprisoned for administering traditional remedies.

According to section 42(2)(a) of the Act, no one is allowed to "sell or supply any product for the purpose of a clinical trial unless that person has a product licence and a clinical trial certificate". This makes it very difficult to assess the efficacy of traditional remedies without following the standard procedures. However, a number of modern medical practitioners have tested the efficacy of some traditional remedies used in Malawi.

Swaziland

There is no government regulation on the use and manufacture of traditional remedies. Modern drugs require registration. Traditional practitioners have been registered since 1974. A list of traditional practitioners is kept by the Swazi National Council, a traditional executive body under the King. In 1981 a Commission for Traditional Medicine was formed by the Minister of Health. The Commission was to recommend ways of organizing the regulation of traditional practitioners and their work as well as to act as a body through which their views are communicated to the government and to the general public.

Tanzania

The legal status of traditional medicine in Tanzania is governed by two statutes namely:

(i) Medical Practitioners and Dentist Ordinance Act, caption 409, section 37, and

(ii) Pharmaceutical and Poisons Act 1978, stipulating that substances used in local systems of therapeutics should be utilized in the communities where "the traditional practitioners belong, provided they are not detrimental to the people's lives and health".

The traditional practitioner is registered by a regional or district cultural officer and his drugs are only known to him or herself. The drugs are not registered. Modern drugs are regulated by law.

Zambia

There are no laws prohibiting the practice and use of traditional medicine. However, traditional practitioners must be registered at provincial level and must adhere to laws governing the practice of modern medicines. There is no regulation in respect of the use of traditional remedies.

Zimbabwe

The government has instituted controls over the practice of traditional medicine through the Traditional Medical Practitioners Act 1981. This made provisions for the formation of a Traditional Medical Practitioners Council and the registration of practitioners. An Association of Traditional Practitioners was formed in 1980. It promotes professionalization and gives direction and support to member practitioners.

There is no drug regulation specifically applicable to traditional remedies. Modern drugs circulating in the country must be registered under the Drugs and Allied Substances Control Act (Chapter 320) 1949.

Conclusion

In all of the participating countries, the general feeling is that the future of traditional medicine is bright, because it is widely used and respected, especially by the rural population that constitute the majority. Although no specific studies have been made, costs are considered to be low.

Legislation is needed in all of the countries to recognize and legitimize traditional practitioners. The traditional practitioners should group themselves into associations through which they could interface with the formal system, whether or not they are formally part of it. An association of this nature could be a regulatory body in relation to ethical and professional matters. Without this formal structure, the chaos that exists now is likely to continue.

Steps need to be taken to list the herbal remedies used in each country and their medical indications and properties. This needs to be done before the disappearance of indigenous people, who hold the key to identifying medicinal plants that may result in new drugs of inestimable benefit to the global community. The establishment of their safety, based on published data and/or preclinical scientific studies, should precede the use of manufactured medicinal plants for both self-medication and in national health services. When quality control has been assured, studies for efficacy may then be initiated.

While these are not unrealizable goals, their attainment will require the establishment of an organizational structure that is coupled with dedication and rational analysis of the situation in each country.

Many African countries are focusing on actions at national level that seek to obtain maximum benefit from their natural plant resources. However, medicinal plants should not be valued solely because of the possibility that they offer from import substitution, but because traditional medicine is an avenue to greater self-reliance, based on appropriate technology in accordance with a country's cultural heritage and national resources. As African countries attempt to revitalize and rationalize this heritage, they can look for support from the World Health Organization in their endeavours.

References

Akerele O. (1988) Medicinal Plants and Primary Health Care: An Agenda for Action, Fitoterapia, Volume LIX, No.5, pp. 355-363.

Akerele O., Stott G., Lu Weibo (eds) 1987. The American Journal of Chinese Medicine, Supplement Number 1, The Role of Traditional Medicine in Primary health Care in China.

Bannerman R.H., Burton J., Chen's Wen-Chieh, Traditional Medicine and Health Care Coverage. A reader for health administrators and practitioners.

Djukanovic, V. & Mach, E.P. (eds.) (1975) Alternative Approaches to Meeting Basic Health Needs in Developing Countries: A Joint UNICEF/WHO Study. Geneva, World Health Organization.

Farnsworth, N.R., Akerele, O., Bingel A.S. Soejarto D.D., Zhengang Guo (1985) Medicinal Plants in Therapy, Bulletin of the World health Organization, 63(6): 965-981.

Report of a WHO/DANIDA Inter-country Workshop on the Selection and Use of Traditional Remedies in Primary Health Care, Kadoma, Zimbabwe, 26 June - 6 July 1989 (in press).

World Health Organization. Alma-Ata (1978). Primary Health Care: Report of the International Conference on Primary Health Care, Alma-Ata, USSR, 6.12 September 1978 ("Health for All" series, No. 1).

WHO (1987) Global Medium-Term Programme (Traditional Medicine) covering specific period 1990-1995 (WHO document TRM/MTP/87.1).

A report on the development of a traditional medicine for bronchial asthma

ALUOCH, J.A., KOFI-TSEKPO, W.M.
WAKORI, E.W.T., RUKANGA, G.M. and TOLO F.

Kenya Medical Research Institute
Nairobi, Kenya

ABSTRACT

A traditional medicine for bronchial asthma was identified through interaction with a traditional healer, Mr. Charles Obuya of Rangwe, South Nyanza. The traditional medicine regimen consists of three different liquid preparations:

(1) A cold aqueous root-bark extract used for diagnosing the disease.

(2) An oral liquid medicine for regular treatment, prepared by boiling plant roots in water.

(3) An oral liquid medicine for regular treatment, prepared by boiling plant stem and leaves in raw ghee.

This traditional medicine regimen is said to produce curative effects in very few weeks. Basic ethnomedical information indicated a high potential in this medicine and this led us to take more interest in the investigation. Phytochemical screening of the drug plant materials, revealed the presence of flavonoids, terpenoids, alkaloids and glycosides. Preliminary animal toxicity studies indicate that the medicine is reasonably safe. There is abundant evidence that the medication has a promising therapeutic effect in man and a clinical study is being planned. The steps taken so tar in the development of this traditional medicine for bronchial asthma will be discussed.

Introduction

Since traditional medicine has been shown to have intrinsic utility, it should be promoted and its potential developed for wider use and benefit to mankind (WHO, 1978). In view of this, the Traditional Medicines and Drugs Research Centre of the Kenya Medical Research Institute, has been able to establish some form of dialogue with the traditional healers on an interactive basis. This has enhanced research on traditional medicines to establish their efficacy and safety.

Asthma is a common and important disease, characterized by widespread bronchial obstruction that is reversible either spontaneously or with therapy. Its principal causes seem to be allergy, infectious, irritants and psychological reactions (Heiner, et al 1973). The large number of conventional medicines currently in use for the treatment of bronchial asthma, are only able to control the disease but do not provide a complete cure. It has therefore been found necessary to develop an asthma traditional medicine prepared by Mr. Charles Obuya, which appears to be of very high potential.

The steps taken so far in the development of this traditional medicine for bronchial asthma are discussed below.

Ethnomedical investigations

The traditional medicine for bronchial asthma was identified through interaction with a medicineman, Mr. Charles Obuya during field research. Several visits were made to his clinic to observe the treatment procedures, and the patients treated with the medicine.

The preparation and formulation of the medicines were observed. The traditional medicine regimen consisting of three different liquid preparations was noted to be prepared from three different plant materials. A medicine for diagnosing the disease is prepared by extracting a root bark in cold water. The cold extract is then administered intranasally at a single dose of 5 ml into each nostril. This results in profuse mucous secretion from the lungs. An oral liquid medicine is prepared by boiling plant roots in water, and the extract is administered at a dose of 200 ml twice a day for two months. A second oral medicine is prepared by boiling plant stem and leaves in water and raw ghee. This is also administered at a dose of 200 ml twice a day for two months or more, according to the severity of the disease.

The medicinal plants used to prepare the medicines were collected, and correct botanical information was obtained with the assistance of the botanists at the herbarium of the National Museums, Nairobi.

The research activities in the Institute have created interest in over 100 asthma patients, who have sought assistance from the Institute in order to use this traditional medicine for asthma. Our laboratories, on the other hand took this opportunity to monitor the conditions of these patients and found that all have responded to this treatment regime. The high potential observed with this medication has led us to take more interest in the investigations .

Phytochemistry

Phytochemical investigations of the plant materials carried out using thin layer chromatography revealed the presence of flavonoids, terpenoids, alkaloids and glycosides.

Pharmacology and toxicology

Preliminary animal toxicity studies were carried out in mice, and the results obtained indicated that the medicine is reasonably safe.

Isolated tissue experiments carried out using guinea pig tracheal rings revealed some antagonistic effects of one of the asthma preparations on the contractions caused by PGF2X.

Clinical perspectives

The therapeutic claims of this medicine were first evaluated by observing the patients under treatment by Mr. Charles Obuya. The medicineman was then invited to our laboratories to carry out a clinical demonstration under the supervision of two physicians among members of the research team. Long function tests were carried out on the patients before and during treatment with the traditional medicine. A reversal of bronchoconstriction was noted on administration of the traditional medicine (Aluoch et al, 1987), indicating a reasonable level of efficacy. Thus there is abundant evidence that this medication is good and a clinical study is being planned.

Discussion and conclusion

In the context of cultural evolution, traditional medicine has always developed and preserved its role of providing care in all communities (WHO, 1978). Thus even if the active principles have not yet been identified in the plants used in traditional medicine, historical evidence of the value of such plants could result in useful preparations provided they are safe (Farnsworth, et al. 1985). The evaluation of chronic toxicity based on the ethnomedical information obtained from the traditional healer and acute toxicity investigated using laboratory mice, suggested that this asthma medication is reasonably safe. The only side effect observed so far is diarrhoea obtained with the use of the oral preparation boiled in raw ghee and water, but this is eliminated by reducing the dose of this medicine.

There are several possible mechanisms which might account for the anti-asthma effect of this traditional medicine. The presence of terpenoids as revealed by the phytochemical screening, may suggest corticosteroid-like mechanisms, e.g., inhibition of histamine formation or storage and the direct smooth muscle effect of steroids. The pharmacological experiments carried out on guinea pig tracheal ring, seems to suggest a prostaglandin pathway as another possible mechanism of action. Further evaluations of these medicines are in progress.

Special tribute

We pay a special tribute to the medicineman, Mr. Charles Obuya for his interest in our collaboration.

References

Aluoch, J.A., Kofi-Tsekpo, W.M., Were, J.B.O., Oyuga, Wakori, E.K., Nganga, L.W. and Obuya, C.O., (1987). In: Kinoti, S.N., Waiyaki, P.G., Were, J.B.O. (eds) Proc. 8th Annual Med. Sci. Conf. Nairobi, Kenya, p. 344-349.

Farnsworth, N.R. Akerele, O., Bignel, A.S., Soejarto, D.D. and Guo, Z. (1985): Bull. WHO, 63(6): 965-981.

Heiner, D.D., Tashkin, D.P. and Whipp, B.J. (1973): Ann. Inter. Med. 78: 405-419.

WHO (1978): The promotion and development of traditional medicine. Technical Report Series 622, Geneva.

Resume of current research in medicinal plants in Botswana

J. BACON

Chemistry Department
University of Botswana
P/Bag 0022 Gaborone, Botswana

ABSTRACT

The potential for the economic development of medicinal plants use in Botswana has been shown to be very great. Experience gained during the last decade shows the necessity for proper management of resources, and a coherent unified strategy for research to reduce the possibility of exploitation of resources by external concerns. The grapple plant, Harpogophytum procumbens, serves as an excellent example of economic exploitation which has necessitated nationwide cooperation of research and government bodies. Following the lessons learned from the grapple plant, traditional remedies are now being closely examined with a more unified approach. Initially, only medicinal plants that have an immediate economic potential are being studied.

Introduction

In common with all African countries, Botswana has a strong tradition in the use of herbal remedies. As is frequently the case, it is difficult to separate traditional religion from therapeutic properties of administered medicines. The value of any drug is greatly enhanced by the power of suggestion, with the conclusion that any innocuous substance administered under the right conditions of suggestion and belief, can have dramatic healing effects. Belief in the power of a drug is not however, limited to traditional medicine. Clinical trials using placebos will always result in a percentage of cases responding to the "drug". For this reason, it is extremely difficult to study possible medicinal properties of plant species and correlate findings with traditional uses. This is clearly exemplified by the "grapple plant" (Harpogophytum procumbens), which, in recent years, has become Botswana's pre-eminent medicinal plant, known in Europe and the USA as "Devils Claw".

In this paper, the author describes the status of the art with respect to the exploitation of the grapple plant and the herbal tea plant (lippia) in Botswana, for medicinal applications.

The grapple plant

The grapple plant grows only under the semi-arid conditions, and is indigenous in the Kalahari desert and parts of Namibia and Angola. It is a typical desert plant in that it shows adaptation to restricted and sporadic rainfall. Much of the plant mass lies below ground level in the form of a parent tuber, storage tubers and roots. The leaf system is highly susceptible to available water, and in times of drought (which is frequent in the Kalahari) may be inconspicuous, making the plant very difficult to identify or collect. The fruiting body has an endocarp which resembles a grappling hook, from which the plant takes its common name. The storage tubers of the grapple plant have been known in Botswana traditional medicine for generations. However, in Namibia, the plant has almost become extinct, due to systematic destruction by the Namibian farmers. The fruiting body can inflict serious damage to animals, and farmers in Namibia regarded it a menace. Its survival in Botswana is probably explained by its use in traditional medicine, and hence its destruction a taboo.

Studies conducted in 1986 by Kgathi, confirm that the grapple was used in small amounts in traditional medicine. Producers of grapple for the European trade, confirmed that it could be used for stomach disorders in man and to heal wounds in animals. However, according to Taylor (1982), clinical trials in Germany indicated that 60% of arthritis cases can be healed by an extract of the grapple storage tubers, with no observable side effects, apart from the purgative effect. It therefore seems apparent, that traditional medicine has utilized the grapple for its purgative effect rather than for its proven anti-arthritic properties. One reason for this may be that the purgative effect is almost spontaneous, whereas the anti-arthritic properties are discerned over a much longer period of time. If this is indeed the case, then the converse must also be true, i.e., detrimental effects of medicinal plants may not be immediately obvious, such that physiological damage may occur days, weeks, or months after receiving treatment. Western medicine has of course learnt this the hard way as in the case of the drug "Thalidomide".

Although the exact mechanism of the therapeutic action of grapple on arthritic cases is not known, the active components of the storage tubers were identified as far back as 1962, by Lux and Tinmann, who identified iridoid glucosides. Bendul et al., (1979) modified the structure to produce an improved form, procumbide. In 1981, Vanhelen et al., proposed a mechanism for the anti-arthritic properties in which they suggested a conversion from harpogoside to harpagogenine. Research is still continuing in Germany as to the exact mechanism involved with these substances.

The case history of the economical development of the grapple plant serves as an excellent example of beneficial exploitation of natural resources and also possible detrimental exploitation of human resources. During the early 1980's 15-20 tones of dried grapple storage tubers were exported yearly from Botswana to Europe, mainly by Namibian and South African traders.

Iridoid glucosides

In 1987, the National Institute of Research (NIR), concluded that in general, producers of grapple are poor people, and in the Kgalagadi district, only those who desperately needed cash were involved in grapple production, because they needed the cash to purchase their basic needs. However, the report also concluded that although the grapple was being produced as a cash crop, it appeared not to have a detrimental effect on production of subsistence crops and the farming activities. The main reason for this seems to be that harvesting of the grapple takes place during the dry season when subsistence crop production has virtually ceased. It is however, interesting to note that the report found that the majority of grapple producers were women, the socio-economic implications of which need to be examined.

In 1981/1982, the average income earned by a grapple producer in the Kgalagadi District was 97 pula. Even allowing for inflation, this sum is small, but the report concluded that it was significant, particularly if it was used for purchasing such basic needs as food and health care.

The economics involved in the grapple trade, are, at best bewildering, and show the need for legislation. It has been calculated (Kgathi, 1987) that one harvester can collect kilogramme of dry grapple in 6.5 hours for which he receives 2 pula, which, although very small, is comparable with the rate for farm workers. It is nevertheless below the minimum wage for manual workers. Both collectors and traders in grapple require permits, and to ensure sustained yields, a quota system is in operation. In order to sell the grapple to foreign traders, the local traders must have an export permit. On the export permit, the amount and value of the grapple is recorded. However, serious discrepancies between the amount bought from producers and the amount exported have occurred in recent years.

The 1987 NIR report notes that although the export prices are recorded in the export permit, they do not make sense, since they are almost equivalent to the prices at which the trader buys from the producers. The report concluded that the correct prices are not actually declared. According to Taylor (1982), a South African company was prepared to pay 4.50 pula per kg for dried grapple storage tubers. Allowing for inflation, this price can now be expected to be much higher.

In 1982 grapple tablets were on sale in U.S.A. and South Africa, at an average price of 5.60 pula per kg (Taylor, 1982). In 1987 grapple tablets manufactured in Europe were on sale, in Botswana, at 148.25 pula per kg. (Kgathi, 1987). In February 1990, the price is 213.25 pula per kg. There is no evidence to suggest that other ingredients are added to the tablets, suggesting that the dried tubers are simply sterilized and compressed into tablet form. Kgathi (1987) concludes that the difference between trader prices and producer prices is just too wide, even if one allows for transport costs. The report recommends that the government should look into this matter and work out possibilities for increasing the producer prices of grapple. It is also apparent that strategies should be developed to lessen the difference between the trader prices and the tablet manufacturers prices.

In 1989, a non-profit making organization for rural development (Thusano Lefatsheng) approached the Ministry of Agriculture for funds to develop marketing and sustained production of grapple in Botswana. Thusano is a commercial concern, involved in the development of Botswana's natural products. Profits from the company are ploughed back into rural development. Research within Thusano liaises closely with many institutions, including NIR/Agricultural Research Institutions and the University of Botswana, Chemistry Department. Thusano's involvement with the grapple plant has so far been restricted to research on sustained yields and some sale of the product to European markets.

Following discussions with representatives from the Ministry of Agriculture, an advisory committee has been set up by the Ministry, with representatives from various institutions involved in natural product research, parastatals and Ministry of finance. In principle, it has been concluded that the research operations of the various institutions should be coordinated by Thusano, with financial support from the government for the development of veld products.

The immediate aim of Thusano is to start the manufacture of grapple tablets for export. If this can be achieved, Thusano will be able to pay the producers competitive prices for their labour and profits can be re-invested into rural development projects. The primary aim is to remove the control of the marketing of grapple from individuals who do not re-invest in rural development.

The formation of the advisory committee for the development of natural products in Botswana is certainly a step in the right direction. If environmental/economical chemical/agricultural research bodies can coordinate their activities, then repetitions of the abuse of the grapple plant can be avoided. There is no doubt that a coherent research programme coordinated by Thusano will undoubtedly serve rural development far better than ad hoc research in the Chemistry Department of the University of Botswana. Thusano currently has a number of projects under development, and the Chemistry Department of the University of Botswana is actively engaged in research of some of these products.

Lipia javanica

A herbal tea, marketed by Thusano is made from the dried leaves of Lippia javanica. The taste is variously described as that of 'mint' or 'vanilla'. In traditional medicine, the plant has a variety of recorded uses throughout the Southern Africa area. The reported uses of Lippia javanica according to Watt et.al. (1962), are as follows:

Xhosa: infusion of leaf and stem for coughs/colds and bronchial infections: disinfecting anthrax infected meal

Kwema: cough/cold remedy

Tswana: cough/cold remedy

Zulu: "gangergous rectis" measles, urticaria and rashes.

Zimbabwe: blackwater fever, malaria, dysentery.

Masai: red ointment for body decoration.

Lobedu: colds/nasal haemorrhage.

Shangana: cough remedy

Swati: influenza/colds

Nunguoi Bushmen: Malaria

Tswana: Insect repellent/insecticide

Early research concluded that flowering tops from Tanzania contained 0.4% of an oil rich in ocimene. The leaves contain an oil that yields 65 - 70% of a liquid of molecular formula C₁₀H₁₆O, which has an odour of lemons.

Research within the Department of Chemistry, University of Botswana, in conjunction with the Analytical Chemistry Laboratory of Utrecht University in The Netherlands, has shown that the essential oil yields a liquid of formula C₁₀H₁₆O. However, detailed analysis using various separation techniques and hyphenated techniques such as C₁₀H₁₆O and GC-F.T. etc., show the presence of three compounds of formula C₁₀H₁₆O.

The major component is 3,7-dimethyl-1,3-octadien-5-one, which is a monoterpene with two geometrical isomers as shown:

Figure

These compounds have previously been identified in Tagetes species, specifically, in Tagetes minuta, from which they take their trivial name Tagetones. The antimicrobial action is being studied by Hethely.

The other compound is also a highly unsaturated ketone with a proposed structure as shown below:

Figure

The decongestant effect of ketonic terpenes is well known (c.f. menthone, etc.) and so it is not surprising that these compounds have a calming effect on respiratory conditions. Similarly, the insect repellant properties of cyclic and acyclic monoterpenes has recently been reported (Wang et al 1985). The anti-microbial properties, however, are rather more difficult to explain on the basis of ketonic structures. However, tagetone exists in equilibrium with the enolic form. This can easily be shown by the temperature dependence of the infrared spectrum. At high temperatures, the carbonyl stretching vibration disappears and a hydroxyl stretching absorption appears instead.

Figure

The formation of an enol may explain the anti-microbial properties since enols are known to show disinfectant properties.

When heated, the above compounds readily polymerize by opening of the double bonds. However, it is suspected that in the case of cis-tagetone, the molecule may also aromatize. This reaction is also possible in the presence of ultra violet light.

The product, thymol, is of course a well known natural product (Thyme oil) and its phenolic nature gives it disinfecting properties.

Figure

The potential use of this plant is very promising. However, we feel sure that much of the chemical analysis may be a replication of work that has already been done and unpublished and/or is under investigation in other regional laboratories since there is insufficient liaison between the various groups undertaking research in the field of medicinal plants. Effective research to aid development can only be achieved by a coordinated approach, both nationally and internationally. For this reason, current research into medicinal plants is being restricted to plants which have an 'immediate' commercial potential.

Acknowledgements

I am indebted to the fullest cooperation of the following,: Dr. T. Tietema, National Institute of Research, Gaborone; F. Taylor, Veld Products, Gaborone; Thusano Lefatsheng, Gaborone; Prof. J. H. van der Maas, University of Utrecht, The Netherlands and Phillips Laboratories, The Netherlands.

Reference

Kgathi, D.L. (1987). NIR Research notes (24), University of Botswana.

Hwang, Y., Wu, K., Kumamoto, J., Axelroad, H. and Mulla, M.S. (1985). J. Chem. Ecol., 11, 1297-130.

Taylor, F.W. (1982). The Resource and its Commercial Utilization of Veldproducts, Plan No. T.B. 7/14/80-8, Ministry of Commerce and Industry Government Printer, Gaborone.

Watt, J.M. and Breyer-Brandwisk, M.G. (1962). The Medicinal and Poisonous Plants of Southern and Eastern Africa. 2nd Edn., Livingstone.

The use of data from traditional medicine: Tunisian experience

K. BOUKEF
C.N.T.S., Rue Djetel, Dahmar, Tunisia

ABSTRACT

The industrial, technological and social developments in the world have significantly contributed to a situation whereby man has neglected the development of expanded uses of traditional medicines. However, our knowledge on the adverse side effects of some of the modern medicines, the emergence of diseases which are incurable with modern medicines, and adverse economic conditions particularly in the Third World countries, have re-activated interest on the development of traditional medicines for use in health care systems, all over the world. This trend has called for scientific verification of the efficacy and toxity of these medicines. The new advances require thorough ethnobotanical investigations on medicinal plants; on the traditional uses of the plants; and the mode of preparation of the medicines by the traditional healers. This paper discusses the Tunisian experience on the ethnobotanical survey of medicinal plants. The data obtained in these investigations, are compared with those reported in countries neighbouring Tunisia.

Introduction

During the second half of the twentieth century, there has been rapid technological development in the search for new drugs. Third World laboratories have been "invaded" by newer and more efficient equipment to handle the isolation and identification of the active principles of plants. During the same period, computers have radically transformed, not only our working and living habits, but also our way of thinking.

Despite the above changes, it has been noted that there is paradoxically a trend to return to nature, and to "soft" medicine. Currently research is being carried out almost everywhere in the world, to try to rehabilitate traditional medicine.

In the developed countries, research to rehabilitate traditional medicine has mainly been a result of industrial development, which was geared towards production and consumption, but overlooked the dangers of such consumption. An awareness of the fact that the use of some drugs is dangerous, has led to a scenario whereby people want to go back to the roots, or to the use of medicinal plants.

In the Third World, economic factors have had a role to play in the use of medicinal plants. Due to the economic crisis, some countries are trying very hard to reduce the health budget, particularly the cost of drugs, by advocating the use of medicinal plants and other natural resources.

How can the resources of traditional medicine be used in a rational way? To answer this question, five steps must be followed: (a) taking stock of the resources of traditional medicine; (b) studying similarities in neighbouring countries; (c) modernizing the farming techniques of medicinal plants; (d) establishing procedures for the processing, quality control and standards of plant-derived products; and (e) testing the inocuity and efficiency of plant-derived products, including toxicological tests.

We now turn to a more detailed description of the above steps, with special reference to the experience obtained in Tunisia.

Stock-taking of the resources of traditional medicine

A research was carried out using a questionnaire which was distributed to primary and secondary school teachers all over the country. The research enabled the establishment of an inventory of about 1250 plants used in traditional medicine in Tunisia. Further field research was carried out in most of the regions in the country, and this helped to add 191 more plants to the inventory.

Similarities with neighbouring countries

The neighbouring countries selected for the study were Algeria and Morocco. In Algeria, Merabet carried out research in 1982, and in Morocco, Bellakdar edited a book on traditional medicine in Western Sahara in 1978. He came out with a list, of 250 species.

The study by the current author has managed to establish a list of 24 species which are used in the same way in the three countries, and 41 species which have; the same indications in at least two countries. The traditional use of 18 of the species in the inventory corresponds to characteristics which are already known, or which can be shown scientifically.

The second step described above is necessary, as it adds to the field research, and enables the researcher to sort out the plants listed in the inventory.

Modernization of the farming techniques of medicinal plants

The percentage of active principles found in the plant itself can be improved by genetic engineering and agricultural production of the plants. We will quote here an example of the results obtained with Solanum sodomeum L., a source of solasodine, a raw material which can be used for the semisynthesis of steroid hormones. The species was improved through farming techniques, and the percentage of solasodine was increased from 2.2% to 4.2%.

Establishing procedures for the processing, quality control and standards of plant-derived products

In order to maintain quality, rigid standards have to be set for plant-derived products. A law was passed in 1985 to govern the pharmaceutical industry and the different articles relating to the execution of the law are being worked out.

Testing the inocuity and efficiency of plant-derived products, including toxicological tests

Although an inventory of at least 18 plants (whose activity was demonstrated scientifically) was made, this is not always done for most of the plants used in traditional medicine. This motivated the author and his associates to undertake research aiming at testing the activity of some plants.

(a) Anti-bacterial and anti-fungal activity

16 plants were tested against 4 bacteria and 6 fungi species by using the technique of dilution, in a freezing solid environment. Six plants revealed an activity estimated at 5mg/ml, which can compare with the antibiotic, streptomycin, and the antifungal agent, griseofulvin. The six plants were: Pistacia lentiscus, Peganum harmala, Agave americana, Anonis natrix, rubus discolor and Ruta montana.

(b) Plants with cytotoxic activity

22 extracts were tested for their cytotoxic activity. The tests used were those which have been recognized by the C.C.N.S.C., using human cancerous cells (KB), and murine cells. The extract from Pergularia tomentosa was the only one which revealed an activity estimated at DI₅₀ = 20 mg/ml.

(c) Algae used as vermifuge

Alsidium coralinum was tested by HPLC, and kainic acid was found to be present. This acid was isolated by Fuhrman in 1981 from another alga, Digenia simplex, and its vermifuge activity has been demonstrated.

(d) Plants with anti-inflammation activity

Calendula arvensis is used in traditional medicine in Tunisia to treat rheumatism. Several components were isolated and identified, such as amino acids, phenol acids, flavonoids and particularly saponosides. The study on anti-inflammation was carried out using the carragenine test. By measuring levels of hormones such as cortisone and haptoglobin, it was possible to isolate and identify a saponoside, arvensoside "A", which could be the source of this activity.

Discussion and conclusions

The testing of the above activities, and the search for new active principles need great human and material resources. However, we are of the opinion that the best way to carry out and implement successfully a programme which aims at studying the use of traditional cures derived from plants, is to work in an environment which has the following combination of factors:

(a) the use of plant-derived cures must be socially acceptable;

(b) there must be expertise in the agricultural and pharmaceutical fields; and

(c) there must be an industrial infrastructure, which deals with the transformation of traditional collections into scientific formulae, which can be prescribed and administered, according to recognized professional medical practice.

Chemical and pharmacological studies of marketed traditional drugs

MESFIN BOGALE*, B.K. NOAMESI** and ERMIAS DAGNE*

*Department of Chemistry
Faculty of Science, Addis Ababa University
P.O. Box 1176, Addis Ababa, Ethiopia

**Department of Pharmacology
Faculty of Pharmacy
University of Science and Technology
Kumasi, Ghana.

Introduction

Most of the medicaments used in the traditional medicine of Ethiopia, as indeed in many other countries, are of plant origin. These traditional medicines are obtained in most cases from healers. However, the very common medicaments are obtainable from vendors.

In most markets one does not fail to find a corner which could be considered as an "open pharmacy" and where medicinal plant preparations are spread out to attract the attention of customers. Vendors do not usually prescribe as the customers are quite knowledgeable about the type of drug they wish to purchase.

A survey of 19 medicinal plant markets of Central Ethiopia (Kloos et al. 1978) identified over 40 common medicinal plants sold routinely. This survey showed that Ethiopia has a rich medicinal plant resource. The interdisciplinary studies of clinicians, chemists, pharmacists, botanists agronomists and anthropologists is necessary to develop more efficient uses for these potential resources. Table 1 summarises the results of the survey of Kloos et al.

The proper authentication of medicinal plants and identification of the active ingredients, is invaluable in the assessment of the pharmaceutical value of the traditional medicines. Although the usage of most of the marketed traditional drugs does not require special knowledge, there are instances where overdosage leads to toxic effects, particularly in the use of anthelmintics. Pharmacological studies, therefore, help not only to determine efficacy of these traditional preparations, but also to establish required dosages.

In this paper, we report the results of a study on one of the marketed drugs of Ethiopia. In the indigenous system of medicine in Central Ethiopia, the roots of Taverniera abyssinica (Leguminosae) are known in the Amharic language as 'Dingetegna' signifying "medicine for sudden illness'. The roots are chewed to alleviate severe stomach pain and fever.

T. abyssinica is an endemic species occurring in Ethiopia and grows up to 2 m high in bushland or on limestone, at altitudes between 1700 and 2200 m. Taverniera belongs to a relatively small genus containing only 15 species found in arid regions, from Egypt to India (Thulin, 1983). Three other species are also known to occur in Ethiopia.

Phytochemical investigations of the roots have revealed the presence of a number of compounds including the isoflavonoids formononetin, afrormosin and the pterocarpans medicarpin and 4-hydroxymedicarpin (Duddeck et at., 1987). It has also recently been shown that extracts of the roots of this plant exhibit antipyretic and analgesic properties (Dagne et al, 1990).

The present investigation has been undertaken to evaluate the spasmolytic and other pharmacological activities of the extract of this plant, in order to establish an ethnopharmacological basis for its use in traditional medicine.

Materials and methods

Plant material

The plant material used in this study was purchased from the main market in Addis Ababa from traditional medicine vendors. For botanical authentication of the plant material as T. abyssinica and for voucher specimens see Duddeck et al. (1987).

Extraction

The powdered root (100 g) of T. abyssinica was soaked in 75% ethanol in water for 24 hrs. The concentrated extract was further extracted with butanol. The butanol extract was successively refluxed for 20 min. each with ethyl acetate, acetone and ethanol. The ethanol portion was used to test on the different models. The other extracts were devoid of pharmacological activity.

Pharmacological tests

Four experimental models were employed to investigate the effects of the extract: anti-ulcer, antiasthmatic (in vivo), oxytocic (both in vivo and in vitro) and the isolated guinea-pig ileum. The extract was found to have an effect only on the isolated guinea pig ileum.

Isolated guinea-pig ileum

Adult guinea-pigs weighing 250-350 g were used. Heal segments (ca. 2-3 cm long) were taken from the caecal end. The muscle was suspended in warm (37° C) Tyrodes solution aerated with atmospheric air in a 20-ml organ bath. Contractions of the smooth muscle were monitored by means of the Ugo Basile isotonic transducer with 1 g tension and recorded on the Ugo Basile Gemini 7070 two-channel recorder at a chart speed of 5-mm/min. The tissue was allowed to equilibrate in Tyrode's solution for 30 min. Control contractile response were obtained for acetylcholine. A contact time of 30 sec. and time cycle of 3 min. was maintained. The extract was then introduced into 500 ml of Tyrode's solution in different concentrations. Using this solution acetylcholine-induced contractile responses were again elicited after giving 20 min. for the tissue to equilibrate every time a fresh solution containing a higher concentration of the extract was used.

In another set of experiments, the effects of the extract of the contractile response of the ileum to histamine were similarly investigated.

Statistical analysis

The given data represent mean ± S.E.M. and the statistical significance was evaluated by the Student t-test.

Results

The extract produced no changes on the resting tone of the isolated guinea-pig ileum, i.e. neither a spasmogenic action nor a relaxation of smooth muscle was observed at any of the concentrations tested. Acetylcholine at concentrations of 5, 10 and 20 ng/ml produced concentration- dependent contractions of the ileum. The acetylcholine-induced contractions were significantly (p < 0.001) antagonized by the extract at 500 and 800 ng/ml. Fig. 1 illustrates a typical effect of the extract on the ileal response to acetylcholine and the results are presented in Table 1.

In the presence of the extract, maximal responses to acetylcholine could not be reestablished by increasing the concentrations of acetylcholine. Histamine at 10, 20 and 40 ng/ml also contracted the guinea-pig ileum in a concentration-dependent manner. The inhibitory effects of the extract on the isolated guinea-pig ileum contractions to histamine are illustrated in Fig. 2 and the results are presented in Table 2. As was observed for acetylcholine, in the presence of the extract, maximal responses to higher histamine concentrations were also not attained.

Discussion

Spasms of the gastrointestinal tract and gastric hyperacidity contribute to the symptoms of stomachache. In orthodox pharmaceutical preparations, such as, belladonna extracts, containing alkaloids of the atropine type, are often included in formulations for stomach ailments, because of their spasmolytic actions against acetylcholine-induced spasms (Weimer, 1980). Histamine mediates gastric acid secretion, acting through the H receptors and has been shown to be responsible for gastric pain, particularly in ulcers. To antagonize the histamine, gastric activity H receptor antagonist drugs like cimetidine, have been designed (Douglas, 1980).

Our present preliminary pharmacological investigations of T. abyssinica have illustrated the ability of the extract to antagonize the smooth muscle spasmogenic actions of both acetylcholine and histamine, two of the most important spasmagens responsible for hyperactivity of the gastrointestinal tract. The non-attainment of the maximum control response of acetylcholine and histamine in the presence of the extract suggests the non-competitive nature of the antagonism.

The above findings show that the extract of this plant possesses analgesic and antipyretic properties, confirming the significance of this traditional drug in ethno-medicine.

Acknowledgements

This work was supported by a grant from the Swedish Agency for Research Cooperation with Developing Countries (SAREC).

References

Dagne, E., Yenesew, A., Capasso, F., Mascolo, N., Pinto, A. and Autore, G. (1990). Ethiopian Med. J. (in press).

Douglas, W.W. (1980). "Histamine and 5-HT and their antagonists". In Gilman, A.G., Goodman, L.S. and Gilman, A. (Eds), The Pharmacological Basis of Therapeutics, Macmillan Publishing Co., New York.: 609 - 646.

Duddeck, H., Yenesew, A. and Dagne, E. (1987). Bull. chem. Soc. Ethiopia 1: 36-41.

Kloos, H., Tekle, A., Yohannes, L.W., Yosef, A. and Lemma, A. (1978). Ethiopian Med. J. 16: 33-43.

Thulin, M. (1983). Opera Bot. 68 : 186-188.

Weimer, N. (1980). "Atropine, scoplolamine and related anti- muscarinic drugs". In Gilman, A.G., Goodman, L.S. and Gilman, A. (Eds), The Pharmacological Basis of Therapeutics. Macmillan Publishing Co., New York: 120-137.

Fig.1. Typical trace showing the contractile responses of the guinea-pig ileum. 'A' shows control responses induced by ACh 5, 10, 20, 40, 80 and 160 ng/ml and 'B' shows responses of the ileum for ACh 0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 ug/ml in the presence of 500 ug/ml of the extract.

Fig.2. Typical trace showing the contractile responses of the guinea-pig ileum. 'A' shows control responses induced by histamine 5, 10, 20, 40, 80 and 160 ng/ml and 'B' shows responses of the ileum for histamine 0.08, 0.16, 0.32, 0.64, 1.28, 2.56 and 5.12 ug/ml in the presence of 500 ug/ml of the extract.

Table 1: Some traditional medicinal plants marketed in Ethiopia

Plant species	Vernacular name	Plant part	Major use
Hagenia abyssinica	Kosso	Flowers	Taenicide
Embelia schimperi	Enkoko	Fruits	Taenicide
Glinus lotoides	Metere	Seeds	Taenicide
Croton macrostachys	Bisana	Bark	Taenicide
Myrisine africana	Kechemo	Seeds	Taenicide
Cucurbita pepo	Dubba	Seeds	Taenicide
	Arusi kosso	Root	Taenicide
Silen macroselen	Wogert	Root	General Medicine
Echinops sp.	Kabaricho	Root	General Medicine
Ajuga remota	Armagusa		General Medicine
Withania somnifera	Gizawa	Stem	General Medicine
T. abyssinica	Dingetegna	Root	General Medicine
Ruta chalepensis	Tena adam	Leaves/fruit	General Medicine
	Altit	Resin	General Medicine
Leonotis velutina	Ras-kimir	Leaves	General Medicine
Lepidium sativum	Feto	Seeds	General Medicine
Pychnostachys sp.	Famfa	Leaves	General Medicine
Phytolacca dodecandra	Endod	Fruit	General Medicine
Cucumis prophetarum	Yemeder-embway	Hoot	General Medicine
Artemisia afra	Chukun	Stem/leaves	General Medicine
Vernonia amygdalina	Grawa	leaves	General Medicine
Aloe sp	Setret	Leaves	General Medicine
Thymus serrulatus	Tosin	Leaves	Expectorant
Rubus sp.	Enjore	Leaves	Expectorant
Lantana trifolia	Kase	Leaves	Expectorant
Rubia discolor	Encheber	Roots	Expectorant
Ocimum sp.	Dama-Kasseh	Leaves/Stems	Expectorant
	Taibedle	Leaves	Tonic
	Ofgahng	Leaves	Tonic
Myrtus communis	Addes	Leaves	Tonic
Coriandrum sativum	Dembelal	Leaves	Tonic
Cymbopogon citratus	Tej-sar	Leaves	Tonic
Rutex abyssinicus	Mekmeko	Root	Tonic
Foenicalum vulgare	Ariti	Leaves/Stem	Tonic
S. longipendunculata	Etsemenahe	Root	Medicomagical
Lagenaria spp.	Kel	Fruit	Medicomagical
Commiphora sp.	Karbe	Resin	Vulneraries
	Dechemarech	Root	Vulneraries
Verbena officinale	Attuch	Leaves	Digestant
Laggare sp.	Kaskase	Leaves	Digestant

Table 2: Traditional medicinal drugs available at the market of Addis Ababa according to a cursory survey conducted in February 1990.

Plant species	Vernacular name	Plant part	Major use
Cymbopogon citratus	Tej-sar	Leaves	Buda-besheta
Achyranthes aspera	Attuch	Roots	Dysentery
Mytrus communis	Addes	Leaves	Dysentery
Allium cepa	Nech-shenkurt	Bulb	General Medicine
Echinops sp.	Kabaricho	Roots	General Medicine
Lepidium sativum	Fetto	Seeds	General Medicine
Ocimum lamiifolium	Dama-kasseh	Leaves	General Medicine
Silen macrosilen	Wogert	Roots	General Medicine
Withania somnifera	Gizawa	Stem	General Medicine
Impatients tinctoria	Ensosela	Leaves	Rheumatism
Ajuga remota	Armagusa	Leaves	Stomach
Artemisia afra	Chukun	Seeds	Stomach
Artemisia rehan	Arriti	Leaves	Stomach
Ruta chalepensis	Tena-adam	Leaves	Stomach
			seeds
Taverniera abyssinica	Dingetegna	Roots	seeds
Embelia schimperi	Enkoko	Fruit	Taenicide
Ghinus lotoides	Metere	Seeds	Taenicide
Cucurbita pepo	Duba	Seeds
Hagenia abyssinica	Kosso	Flowers	Taenicide
Dovyalis abyssinica	Koshim	----	Wounds
	Senafech	Seeds	---
	Kosseret	---	---
Osyris abyssinica	Kerett	Roots	---

Research into medicinal plants: The Somali experience

ABDULAHI S. ELMI

Department of Pharmacology
Somali National University
Mogadishu (Somalia)

Introduction

Herbal drugs have a considerable use throughout the World. In the past centuries, such use was understandably more extensive when related to the density of the populations. Treatment with herbal drugs seemed to be destined to vanish with the development of biomedicine. Instead, what actually happened is that despite the expeditious and impressive progress of "modern medicine" in the course of this century, ethnomedicine has remained the chief therapeutic reliance for hundreds of millions of people.

People have recourse to herbal drugs for a variety of reasons. A large number of persons depend on medicinal plants, mainly because they have no access to modern medicine. These people mostly live in rural areas, or in peripheral slums of big cities. For some people, especially in economically developed countries, plant-derived drugs are associated with memories of good old days. Nostalgia for grandmother's remedies are an inducement for many to try such remedies. Certain people believe that natural products have great efficacy while being devoid of toxic effects. Some people rely on modern medicine for certain diseases, while for others they resort to traditional medicine.

The use of herbal drugs by many is the result of balanced judgement based upon personal experiences, or acquired through reliable scientific sources. Whatever the reasons behind the utilization of herbal drugs, the merits of this system of treatment is unquestionable. It is unfortunate that some people associate it with the nostalgia of the past or link it with poverty. Herbal drugs are neither the medicines of the poor alone nor the remedies for nostalgic people; they are not merely a great potential for delivering health care for all in the future; they are actually an important tool for treatment of millions of people of different culture, social class and status throughout the world.

In today's world therapeutic year of armamentarium, plant products are well represented. Farnsworth points out that one quarter of the total prescription drugs in industrialized countries contain one or more components derived from plants.

Furthermore, scientific research has very often shown that in spite of being based on empirical systems, traditional herbal remedies are the result of long standing positive experience.

It is time that the experience of so many generations be placed at the service of modern man without loosing time or necessarily making use of expensive and sophisticated methods. The goal of improving and exploiting the use of medicinal plants in health care can be achieved with relatively easy means and in reasonable time.

Herbal drugs in Somalia

Traditional medicine uses different methods for curing diseases. The Somali traditional medicine could be divided into: (a) ceremonial healing and (b) practical treatments and herbalism.

Ceremonial healing:

This system is based on the celebration of specific rites. Some of these are purely religious. Others are located in the sphere of the magical and others are a mixture of both. The magic rites deal often with spirits and the treatments are mainly for mental or psychosomatic disorders. Famous among these rites are: the saar, hayaat, mingis, nuumbi, etc.. The religious treatments are based on the islamic teaching, that is the Koran, and give health to the true Muslim believers. Religious healing is for both organic and psychic diseases.

Practical treatments and herbalism:

These systems deal more properly with organic disorders. Most common among these are: (i) cauterization, (ii) scarification and blood letting, (iii) bone-setting, (iv) surgery, and (v) use of herbs. Traditional medical treatments are well approved and widely used by the Somali population. Surveys on traditional medical practices carried out by the Division of Pharmacology of the Faculty of Medicine in different times, showed very high prevalence of this type of medicine within both the rural and the urban communities. Among other information, one survey indicated that in the male population, the administration of herbs reached 73%. Several hundred plants are used in Somali traditional medicine. The confidence of the population to the ability of traditional herbalists is great. The use of plants is not devoid of spiritual rites. In the Somali traditional medicine, there is a great respect for the plant. Eradication of the whole plant is avoided, even if the used part is the root. This shows also a respect to the environment. Healers of the inter-riverine area do not consider the plant as a simple physical entity. Greater part of herbalists feel that the effect of a plant depends not only on its power, but also on the relationship between the collector and the plant itself. Usually, a healer avoids his shadow on the plant while collecting it. He says prayers or recites formulas before cutting the plant. The recited words or formulas may be words from the Koran or prayers to ancestors. It is important that the rules laid down by the ancestors be strictly followed.

Most herbalists make use of no more than 30-40 different plants. Nevertheless, the average number of plants known to the majority of healers is far greater than that. Many herbalists could easily list over 100 plants, indicating the purpose they are used for in traditional medicine. In this they are like the modern physicians, who in spite of the great armamentarium of drugs at their disposal, feel more convenient to prescribe few dozens of drugs during their lifetime. The average inventory of kinds of leaves, stem barks and roots in Mogadishu traditional herbalists' dispensaries do not exceed the number of 35-40 for each. While in the rural areas healers very often go out into the bush in order to collect their own herbs such is not the case in the cities. The herbalists who are also dispensary owners would employ an apprentice, or younger herbalist for this job. They also buy herbs by occasional suppliers. By doing so, much of the magical aureola is neglected. They prefer to pretend that their suppliers have complied to all traditional plant collecting regulations. Many herbalists of the cities probably do not give great importance to the "rules of the ancestors".

Herbalists of big centres may act as healers or simply as dispensers. In fact they may dispense herbs on simple request by the patient or according to another healer's prescription. This is quite a difference compared to their rural counterparts, who gather herbs upon clients needs. Traditional herbalists are allowed to practice their profession without restrictions. On the other hand, the law is not clear on whether clinical trials with plants could be performed.

Research experience

A programme of research into medicinal plants was established by the Somali National University in 1978. Investigation on plants used in traditional medicine is also one of the main lines of research of the Somali Academy of Sciences and Arts. The aims of the research that started in 1978 are:

(a) to foster the accomplishment of better use of medicinal plants lending to the necessary scientific support;

(b) to examine the credits of traditional use of medicinal plants in the light of modern science so as to encourage the use of therapeutically effective plants and discourage harmful ones;

(c) to promote the integration of proven valuable knowledge in herbal and modern medicine;

(d) to stimulate and cooperate in the realization of Somali traditional pharmacopoeia;

(e) to reduce the country's drug bill;

(f) to help in creating a national pharmaceutical industry;

(g) to aid in the therapeutic, economic and commercial exploitation of medicinal plants, by promoting their use, culture and exportation.

The research is a multi-disciplinary enterprise requiring the contributions of botanists, chemists, pharmacologists, and clinicians. At the Somali national University, the research on medicinal plants involves the Division of Pharmacology, Faculty of Medicine, the Section of Organic Chemistry, Department of Chemistry, and the Division of Botany at the Faculty of Agriculture.

At the very beginning, in 1978, we designed our programme just following the classical approach for drug research. Great importance was given to the isolation and structure elucidation of active compounds and pharmacological screening on them. After sometime, the team of research realized that the system chosen for the research was not the most appropriate to attain the aims of the programme at reasonable time. Further discussions brought about some changes and a decision was made that the research phases be as follows:

(a) Inventory of botanical identification of plants used in traditional medicine.
(b) Literature survey of the identified plants.
(c) Verification of efficacy of selected plants.
(d) Safety and toxicity assessment of active plants.
(e) Isolation, identification or structure alienation of active principles.
(f) In-depth pharmacological and toxicological evaluation of isolated active substances; and
(g) Production of drugs based on plants containing therapeutically valuable substances.

Extensive work has been accomplished on each of the above phases. The plants to be investigated upon are not chosen at random, but according to clearly set priorities. These priorities are linked to:

(i) the prevalence of the use of the plant among the population;
(ii) the prevalence of the disease for which the plant is used. Additionally, plants used for diseases which have no good cures in modern medicine, are given due consideration.

Regarding the inventory and botanical identification, information on the use of hundreds of plants has been collected by interviewing traditional herbalists. Many plant collecting expeditions have been carried out. All the collected plants have been identified. Samples of collected plants have been sent to internationally important herbaria.

Literature information has been collected for a relevant number of plants. This was partially carried out in Somalia. Lists of names (with synonyms) of identified plants were sent to the WHO collaborating Centre for Traditional Medicine at the University of Illinois, Chicago, USA, for search, through the NAPRALERT computer file. Literature printouts for most of the identified plant species have been obtained from the above Centre. The Medicinal Plants News-letter published by OAUSTRC, also reports literature information on medicinal plants.

Following the above system, extensive experimental research through the use of in vivo and in vitro pharmacological methods has been carried out. The performed activities include: isolated organ tests, antimicrobial and antiparasitic activity, anti-inflammatory activity, anti-ulcer activity and several others. Toxicological studies have been performed on a number of plants.

The isolation and identification of active principles has led to the elucidation of the structure of a number of compounds. Some of these compounds, such as, two 1,3-diarylpropan-2-ol derivatives, called quracol A and quracol B, are new compounds hitherto not found in plants. One of the positive results of this chemical research was the identification of a cocancerigenic compound (a phorbol diester) in a plant species, the oil of which was commercially exploited by a Government agency for use as a purgative.

The last step is the clinical evaluation of efficacy and safety. This is the most difficult phase, especially because of the ethical implications and the long time required for carrying out appropriately controlled clinical trials. We elaborated a strategy that would allow us to monitor some clinical effects before starting with controlled clinical trials. Since the traditional medical practitioners are allowed to practice their profession, we decided to assign a physician to a qualified and licenced healer. The healer's job was mainly observation of the healer while he practises. This arrangement was not difficult, because a practicing healer was in fact among the staff of the Division of Pharmacology. The observations yielded valuable information on several plants.

The research programme has given a lot of interesting and useful results. The new approach has shown to be better suited for the aims of the programme. Nonetheless, it has many shortcomings.

The experience has shown that it still neglects the most important and immediate objective of medicinal plants research in a developing country: the early utilization of these plants in Primary Health Care. Most of the research programmes in developing countries share these drawbacks.

More appropriate method for applicable research

The research into herbal drugs usually makes use of dried plants, while we know that such plants are normally administered by traditional medical practitioners in the fresh state. Moreover, the solvent used by the practitioners is water.

The classical method for research is to dry the plant, store it for some time and then subject it to extractions with different types of solvents. Thus the approach of the researcher is quite different from that of the operators of the type of medicine which is under evaluation. It is clear that the researcher directs the work in a way more compatible with the setup of the research facilities and methodologies. The latter are established according to drug research of pure chemical compounds. In fact the rest of the research sequence is testing on laboratory animals and later on clinical trials as is classically done with synthetic drugs.

Is this method appropriate for plant material? Many plants undeniably lose totally or partially their activity during the drying and storing process. Therefore biological as well as chemical studies must be performed on fresh plants. The use of solvents and fractionation may result in greater concentrations of active compounds and stronger activity. But this is not always the case. In fact, sometimes total activity decreases with fractionation.

The classical method gives undue importance to the isolation of pure active compounds from medicinal plants. While isolation and identification of single active compounds is interesting for studies of structure-activity relationships and may be stimulating for the scientist, it will not contribute to any significant extent to the solution of health problems of developing countries. It is imperative that research methodologies be made more respondent to the principles of traditional medicine and to improved objectives. We must consider that traditional medicine has, in many countries, greater prevalence and accessibility than modern medicine. There is no doubt that the trend will remain the same for many years to come.

For the hundreds of millions of people who live in rural areas, changes of attitude and the established use and acceptance of modern health care facilities will be very gradual. Therefore, the immediate useful arid most important contribution of scientists in this field is how to make the traditional curing systems safer and confirm or disprove the efficacy of the preparations which so many people make use of.

If research into medicinal plants is oriented to reach this very important goal, it can be carried out in an easier, quicker and cheaper way, than the methods which are normally applied in most research centres of developing countries. People in our countries are using herbal remedies although for most of them the toxicity has not been studied. It is the duty of scientists to investigate the toxicity of every product which is consumed by humans. One of the first investigations on all medicinal plants, regardless of their efficacy is, therefore, the study of their toxicity.

The second step is the evaluation of the activity for which the plant, or combination of plants, is used. If for nothing else, it is very unwise and wasteful to use something when it does not serve the purpose for which it is used.

Once enough information has been acquired on the safety and efficacy of a certain traditional remedy, this knowledge must be transferred to those who prescribe the treatments and, possibly, to the clients who make use of such treatments. Normally, the results on the investigations of plants remain in the drawers of the laboratories or in libraries as printed materials and they will never reach the user of the plants.

The method that we deem best respondent to the needs of our communities is as follows:

(a) toxicological study in two species of animals for acute and subacute toxicity;

(b) experimental evaluation of the activity for which the supposed remedy is used; and

(c) clinical evaluation for efficacy in humans (where possible this must be preceded by observation of the healer while using the remedy).

The fact that the plant is already used by healers on humans should not, by any means, save it from the necessary ethical obligations during clinical trials.

The advantage of this model is that the costly, sophisticated and time-consuming chemical studies of separation, subsequent fractionations and structure elucidation is avoided. These steps, in fact, are not necessary for the needed progress towards a better use of medicinal plants in health care. This approach takes into account the concepts of traditional and folklore medicine. We cannot expect that traditional medical practitioners make use of pure extracts, or fractions of the plants they use,

The organization of training courses and workshops with the participation of healers would contribute to the improvement of their knowledge and skills and to the consolidation of a safer and more effective community health care system. Healers trained and left to operate in their communities would be the best fabric for Primary Health Care.

The achievement of this goal would be the greatest satisfaction and victory for scientists engaged in research into medicinal plants.

Effect of nitrogen and phosphorus on the essential oil yield and quality of chamomile (Matricaria chamomilla L.) flowers

V.E. EMONGOR*, J.A. CHWEYA*, S.O KEYA* and R.M. MUNAVU**

*Crop Science Department, University of Nairobi
P.O. Box 29053, Nairobi, Kenya

** Department of Chemistry, University of Nairobi
P.O. Box 30197, Nairobi, Kenya

ABSTRACT

Field experiments were carried out to determine the effect of nitrogen (0, 50, 100, and 150kg N/ha) and phosphorus (0, 17.47, 34.93, and 52.41 kg P/ha) and their interactions on the essential oil yield and composition of chamomile. Nitrogen significantly increased essential oil yield and influenced its composition. Phosphorus did not significantly influence essential oil yield and composition, but low phosphorus rates (17.47 kg P/ha) tended to increase essential oil yield. High phosphorus rates decreased essential oil yield. Application of 17.47 kg P/ha at transplanting and top-dressing later with 50 kg N/ha gave the best results.

Introduction

Chamomile flowers contain an essential oil which is used in the manufacture of drugs for the treatment of such diseases as convulsions in children, diarrhoea, colic and acidity, hysteria, allergy, inflammation of body tissues, sleeplessness and stomach ulcers induced by chemical stress or heat coagulation (Martindale, 1977; Sticher, 1977 and Isaac, 1980). The essential oil also promotes epithelization and granulation, and shows antibacterial and antimycotic effects, through the activity of (-)-a-bisabolol and chamazulene (Isaac, 1979). The oil can also be used for flavouring liquors, colouring foods and making cosmetics (Bailey, 1949 and Kirk and Othmer, 1952). The essential oil content of chamomile flowers is in the range of 0.2-2.0% per unit dry flower weight (Martindale, 1977 and Franz, 1980). The composition and yield of essential oil may be affected by many factors, including plant nutrition (Franz et al., 1978 and Franz. 1982).

Work done elsewhere, and not in Kenya, has shown that nitrogen and phosphorus fertilization increases the yield and essential oil content of the flowers (El-Hamidi et al., 1965; Franz 1981; Singh, 1977 and Meawad et al. 1984). The authors further reported that nitrogen and phosphorus influenced oil composition. Although nitrogen and phosphorus increased chamazulene content in the essential oil, excess nitrogen decreased it. Franz (1983) reported that nitrogen increased the concentration of (-)-a-bisabolol but decreased that of bisabololoxide B. No work on chamomile has been conducted in Kenya.

The importance and usefulness of chamomile essential oil in the pharmaceutical, food, and cosmetics industries and the fact that Kenya is importing a lot of the essential oil, has led to the initiation of studies on chamomile. The objective of this study was to show the effect of nitrogen and phosphorus and their interactions on the essential oil yield and composition of chamomile flowers.

Materials and methods

Field experiments were carried out between August, 1985 and March, 1987 at the Field Station, Faculty of Agriculture, University of Nairobi. Chamomile seeds (variety max et oljea) were sown in the nursery and seedlings were transplanted four weeks after germination, when they had attained 6-7 true leaves. The treatments consisted of 4 levels each of phosphorus (0, 17, 47, 34.93 and 52.41 kg P/ha) and nitrogen (0, 50, 100, and 150 kg N/ha). These were combined factorially to give 16 treatment combinations which were laid down in a split-plot design with three replicates. Phosphorus and nitrogen treatments were allocated to main plots and sub-plots, respectively. Phosphorus and nitrogen were applied at transplanting time and two weeks after transplanting, respectively.

Harvesting of flowers started when 50% of the plants had flowered and continued for 98 days. At every harvest, only flower heads with more than 40% open tubular florets were harvested. The fresh flowers were dried to constant weight in an air-ventilated oven, at 35° C for 5 days and their dry weights were then determined and cumulated. The cumulated dry flowers were then used for extraction in order to determine the quantity and quality of the essential oil.

Determination of the quantity of the essential oil in the dried flowers was based on steam distillation. Clevinger apparatus were used for the extraction using the method described by Trease and Evans (1978) and Kornhauser (1986).

The qualitative analysis of the essential oil was done using gas liquid chromatography (GLC) as outlined by Kirk and Othmer (1952), Trease and Evans (1978) and Kornhauser (1986), with slight modifications on the conditions of the GLC. The conditions of the GLC used were as follows: Apparatus: Gow-mac series 69-750; column: 2.5 m long, 0.25 cm internal diameter; Packing: OV-1 on chromosorb W/HP (100-120); Temperature linear programming, 85- 175°C, 2.5°C per minute; Detector: Flame ionization; Injector temperature: 220°C; Detector temperature: 220°C; Column temperature: 170°C; Carrier gas: Nitrogen (flow rate 25 cm³ per minute); Attenuation: 16; Chart speed: 1 cm per minute; and Range: 10^-11. The results presented are means of two trials.

Results discussions

Essential oil yield

Nitrogen fertilization significantly increased essential oil yield per both unit dry flower weight and hectare (Table 1). Increasing nitrogen from 0 to 100 kg N/ha increased essential oil yield per both unit dry flower weight and hectare from 0.627 to 1.036% (65% increase), and 5.85 to 16.64 kg (184% increase), respectively. Nitrogen rate above 100 kg N/ha decreased oil yield. Similar results were reported by El-Hamidi et at., (1965), Franz (1981), Agena (1974), Meawad, (1981) and Meawad et al. (1984); that is nitrogen increased chamomile essential oil content and yield. The increase of essential oil yield due to nitrogen fertilization could be accounted for by the fact that nitrogen played an active role in the development and division of new essential oil cells, cavities, secretory ducts and glandular hairs (Meawad 1981; Meawad et al, 1984 and Agena, 1974). Nitrogen may have increased the essential oil yield because of increased carbohydrate accumulation, gibberellins and auxins concentration in chamomile plants. These were then utilised in the formation of more essential oil cells in the secretory ducts, cavities or glandular hairs (Sacks and Kofranek, 1963; Moore, 1979; Agena, 1974 and Abou-Zeid and El-Sherbeeny, 1974).

Table 1: Effect of nitrogen on essential oil yield of chamomile plants

N rates kg N/ha	Essential oil yield per unit dry flower weight*	Essential oil yield per plant (Kg/ha)
0	0.627a	5.85a
50	0.869c	13.08b
100	1.036d	16.64b
150	0.811b	13.16b

* These values are ratios and hence they have no units

Effects of phosphorus and nitrogen and phosphorus interactions on essential oil yield per both unit dry flower weight and hectare were not significant.

Essential oil composition

Nitrogen fertilization significantly increased chamazulene, (-)-a-bisabolol and farnesene concentrations in the essential oil of the flowers (Table 2). Increasing nitrogen from 0 to 50 kg N/ha increased chamazulene, bisabolol, farnesene and cis-spiroether contents by 25, 13, 11 and 15%, respectively. Application of nitrogen above 50 kg N/ha led to a decrease in the contents of these constituents. However, bisabolol content increased throughout with increase in nitrogen. Similar results have been reported by Agena (1974), Franz (1981) and Franz (1983). The increase of chamazulene (matricine), bisabolol, farnesene, and cis-spiroether concentrations in the essential oil of chamomile flowers with increase in nitrogen application could be due to the decrease in the contents of bisabololoxides A and B with increasing nitrogen application. Amino acid metabolism in nitrogen-rich chamomile plants leads to the biosynthesis of chamazulene (matricine), bisabolol, farnesene and cis-spiroether at the expense of bisabololoxides A and B and vice versa (Franz, 1981 and 1983). This implies that the biosynthesis of basic hydrocarbon terpenes (matricine, farnesene and bisabolol) of chamomile are antagonistic to that of the oxygenated terpenes (bisabololoxides and bisabolonoxides).

Nitrogen application significantly decreased the concentrations of both bisabololoxides A and B in the essential oil of the flower (Table 2). Increasing nitrogen from 0 to 150 kg N/ha resulted in a decrease of 27 and 39% in bisabololoxides A and B concentrations, respectively. Franz (1981 and 1983) reported similar results.

Bisabololoxides (A + B) were predominant in the essential oil of the flowers, as they constituted on the average, 54.21% of the total constituents. Other constituents included bisabolol 6.02%, chamazulene 7.76% farnesene, 13.65% and cis-spiroether 7.97%. Mr-lianova and Felklova (1983) reported similar results. They reported that bisabololoxides (A + B) contents in essential oil of chamomile flowers were over 50%. This can be attributed to the fact that the biosynthesis of bisabololoxide A and B, and bisabolol are controlled by dominant and recessive genes, respectively (Franz, 1982).

Phosphorus application and the interaction between nitrogen and phosphorus did not significantly influence essential oil composition of chamomile flowers.

Table 2: Effect of nitrogen on essential oil composition of chamomile flowers

N rates kg N/ha	% chamazulene	% bisabolol	% farnesene	% cis-spiro ether	% bisabololoxide A	% bisabololoxide B
0	6.89a	5.17a	12.93a	7.38a	43.55d	22.69d
50	8.60c	5.84b	14.31b	8.46a	38.68c	18.11c
100	8.02bc	6.51c	13.84ab	8.13a	34.82b	15.09b
150	7.45ab	6.54c	13.90ab	7.90a	31.58a	13.20a

Figures followed by same letter(s) down the columns are not significantly different according to Duncan's multiple range test at 5% probability level.

Conclusion and recommendation

The study showed that application of 17.47 kg P/ha (40 Kg phosphorus pentoxide, P₂O₅/ha) during transplanting and two weeks later top-dressing with 50 kg N/ha, would ensure high essential oil yield which has good quality. The study also showed that nitrogen was important in the biosynthesis of essential oil and its components. However, it is recommended that more research should be done in the field of plant breeding, agronomy (varietal evaluation, plant nutrition, ecological zones), plant biochemistry and economic evaluation of chamomile growing in Kenya.

Acknowledgements

The authors are grateful to the University of Nairobi for financial assistance during the period of this study. They also wish to record their thanks to Dr. B.O. Mochoge of the Department of Soil Science for his assistance during the laboratory work.

References

Abou-Zeid, E.N. and El-Sherbeeny, S.S. (1974): A preliminary study on the effect of GA on quality of volatile oil of Matricaria chamomilla L., Egypt J. Physiol. Sci. 1: 63-70.

Agena, E.A. (1974): Effect of some environmental and soil factors on growth and oil production of chamomile (Matricaria chamomilla L.). Ph.D. thesis, Faculty of Agriculture, Ain Shams University, Egypt.

Bailey, L.H. (1949): Manual of cultivated plants, Macmillan Publishing Co. Inc., New York: 99-991.

El-Hamidi, A. Saley, M. and Hamidi, H. (1965): The effects of fertilizer levels on growth, yield and oil production of Matricaria chamomilla L. Lloydia 28: 245-251.

Franz, C. Holzl, J. and Vomel, A. (1978): Variation in the essential oil of Matricaria chamomilla L. depending on plant age and stage of development. Acta Hort. 73: 229-238.

Franz, C. (1980): Content and composition of the essential oil in flower heads of Matricaria chamomilla L. during its ontogenetical development. Acta Hort. 96: 317-321.

Franz, C. (1981): Zur Quabitation arznei and Gewurzplanzen Habilschrift Tumuchen: 280 Habilitations Schrift, Weinhenstephan: 301-307.

Franz, C. (1982): Genetic, ontogenetic and environmental variability of the constituents of chamomile oil from Chamomilla recutita, Freising-Weinhestephan D-8050/F.R.G.: 299-317.

Franz, C. (1983): Nutrient and water management of medicinal and aromatic plants. Acta Hort. 132: 203-215.

Isaac, O. (1980): Antibacterial and antimycotic effects of bisabolol. Dtsch. Zeg. 120: 567.

Kirk, R. E. and Othmer, D.F. (1952): Encyclopedia of chemical technology 9: 569-591.

Kornhauser, A. (1986): UNESCO University-Industry Co-operation Project on Matricaria chamomilla L., Seminar/Workshop Nairobi. Kenya.

Martindale, W. (1977): The extra pharmacopoeia, 27th edition: 1011- 1021.

Meawad, A. A. (1981): Physiological and anatomical study on gladiolus. Ph.D. thesis, Faculty of Agriculture, Zagazig University, Egypt.

Meawad, A. A., Awad, A. E. and Afify, A. (1984). The effect of nitrogen fertilization and some growth regulators on chamomile plants. Acta Hort. 144: 123-134.

Moore, T.C. (1979): Biochemistry and physiology of plant hormones, Springer Verlag Inc., New York, U.S.A.: 90-142.

Mrlianova, M. and Ferklova, M. (1983): Content of bisabololoxides in flower heads of Matricaria chamomilla L., Farm obz. 52: 257-266.

Sacks, R. M. and Kofranek, A. M. (1963): Comparative cytohistological studies on inhibition and promotion of stem growth in Chrysanthemum morifolium. Amer. J. Bot. 50: 772-779.

Singh, B. (1977): Cultivation and utilisation of mediana (Matricaria chamomilla L.) and aromatic plants in Afal and Kapur, India, RRL. Jammu-Tawi: 350-352.

Sticher, O. (1977). New natural products and plant drugs with pharmacological, biological or therapeutical activity, Proc. 1st Internat. Congress Med. Plants Res., Sec. A., Univ. Munich, Germany: 136-176.

Trease, G. E. and Evans, W. E. (1978): Pharmacognosy. 11th edition, Bailliere, Tindall, London: 255-281, 405-474.

Chemical characterization of pharmacologically active compounds from Synadenium pereskiifolium

KERSTIN HERMANSSON*
LENNARD KENNE*, GEOFREY M. RUKUNGA**
GUNNAR SAMUELSSON*** and W. M. KOFI-TSEKPO**.

* Department of Organic Chemistry, Arrhenius Laboratory,
University of Stockholm, S-106 91 Stockholm, Sweden.

** Kenya Medical Research Institute
Traditional Medicines and Drugs Research Centre
P.O. Box 54840, Nairobi, Kenya.

*** Department of Pharmacognosy
University of Uppsala, Biomedicum
P.O. Box 579, S-751 Uppsala, Sweden.

ABSTRACT

Synadenium pereskiifolium (Baill.) Guill (Euphorbiaceae) is the key plant among the six plants which are used in the preparation of an anti- asthmatic drug regimen by traditional doctors. Although this plant belongs to the family of poisonous plants, traditional doctors have used it effectively in the treatment of asthma for decades with no adverse effects. Phytochemical screening of the aqueous extract of this plant revealed the presence of glycosides, terpenoids, flavonoids and other phenolic compounds. In order to characterize the pharmacologically active compounds from the aqueous extract of S. pereskiifolium, a method was adopted that was based on ion exchange, gel nitration on sephadex and extraction with organic solvents.

Introduction

Synadenium pereskiifolium (Baill.) Guill, belongs to the family Euphorbiaceae. The plant is used in the preparation of various traditional medicines, the most important preparations being an asthma remedy. S. pereskiifolium has been reported in the literature (Verdicourt and Trump, 1969; Watt and Breyer-Brandwijk, 1962) as a poisonous plant and no therapeutic value has to-date been ascribed to it. There are several publications which have mentioned other plants used traditionally for the treatment of asthma (Adjanohoun, 1983; Oliver, 1960; Nad Karni, 1976; Kokwaro, 1976; Watt and Breyer-Brandwijk, 1962). None of the publications have mentioned S. pereskiifolium as a drug plant for asthma. Yet in the preliminary study in our laboratories, medicines prepared from this plant by a traditional medicineman have shown very promising therapeutic effects on man. Preliminary phytochemical screening revealed that the leaves and stems of the plant contained glycosides, flavonoids and terpenoids.

An aqueous extract of the stems and leaves of S. pereskiifolium showed both contracting and inhibition activity of the isolated Guinea pig ileum. The aqueous extract was thus subjected to a bioassay-guided fractionation according to the scheme for preliminary chemical characterization of pharmacologically active compounds in aqueous plant extracts (Samuelsson et al., 1985).

Experimental

Solutions were concentrated under reduced pressure at temperatures not exceeding 40°C. Proton nuclear magnetic resonance (¹H-NMR) spectra were obtained at 270 MHz, and Carbon-13 nuclear magnetic resonance (¹³C-NMR) spectra were taken at 67.8 MHz on a JOEL GSX-270 spectrometer using sodium 3-trimethysilyl-propanoate-d₄ i (TSP, ¹H-NMR, D₂O) and 1, 4-dioxane (¹³C-NMR, D₂O; 67.40) as internal references. Spectra were obtained at 70°C. Separation of 2-butyl glucosides was performed on Hp-54 fused-silica capillary columns (30 m × 0.3 mm) at 190-250°C, 3°/min. A Hewlett Packard 5970 MSD gas chromatograph - mass spectrometer (GC-MS) was used for GC-MS analysis. Positive FAB-MS spectra were obtained on a JEOL Dx-303 spectrometer.

Plant material

Fresh aerial parts of S. pereskiifolium were collected from South Nyanza, Kenya and transported to Sweden by airfreight. The identity of the plant was established by Dr. Mats Thulin, Department of Systematic Botany, University of Uppsala, Sweden.

Extraction

The fresh material (1.2 kg) was cut to small pieces in a blender with rotating knives and extracted in water (81) by stirring at room temperature overnight. The extract was filtered, concentrated in vacuo in a cyclone evaporator and lyophilized, yielding crude material (41.9 g).

Isolation of glucosides

Crude extract (10 g) was dissolved in water (100 ml) and acetone (1 l) was added with stirring. The precipitate which formed was recovered and lyophilized, yielding 8.0 g of material. An aqueous solution of this material was applied on Dowex 50 (H⁺) (160 ml) and eluted with water until the effluent was colourless. The eluate was neutralized with ammonia, concentrated in vacuo and lyophilized, yielding 5.0 g of material. The water eluate was partitioned between water (300 ml) and n-butanol (5x200 ml). The aqueous phase was concentrated in vacuo and lyophilized, yielding 4.8g of material. Part of this material (2.5g) was subjected to flash chromatography on silica gel (180 g) eluating with methanol: acetic acid: chloroform (85:10:5v/v). The separation was monitored by thin layer chromatography (TLC) using ethanol: acetic acid: propanol (50:30:10 v/v) and the compounds were visualized by spraying with anisaldehyde-sulphuric acid. One fraction contained a component which gave a green spot on TLC. The solvent from this fraction was evaporated and the material was lyophilized (0.9 g). Further purification of the material (100 g) was performed on Sephadex LH 20. Eluation was performed with water and the separation was monitored by TLC. Fractions containing the compound giving a green spot were combined and lyophilized (56 mg). The yield corresponded to 8.9% of the original aqueous extract of the plant and 0.3% of the fresh plant material. Part of the material was transformed to the acid form by passing it through Dowex 50 (H⁺). The sodium salt was obtained by evaporating part of the material with sodium bicarbonate followed by purification on a column of Bio-gel P-2. The material was analysed by MS and NMR spectroscopy.

Figure

Acid hydrolysis of the glucoside

The glucoside (43 mg) was treated with 2M trifluoroacetic acid for two hours at 120°C. The reaction mixture was purified over Bio-Gel P-2, eluted with water. A fraction containing pure aglycone was obtained and the latter was shown to be malic acid by NMR and MS spectroscopy and comparison with authentic L-malic acid. Glucose was also isolated from the reaction mixture and identified by sugar analysis and ¹H-NMR spectoscopy.

Determination of the absolute configuration

The glucose (2.7 mg) was treated with 2M hydrochloric acid in (+)- 2- butanol (0.2 ml) at 80° for eight hours in a sealed tube (Gerwig, et al, 1978). The mixture was neutralized with silver carbonate and then evaporated to dryness over phosphorus pentoxide. Part of the material was analysed by GC-MS and another part was silylated with a mixture of trimethylchlorosilane-hexamethydisilane (1:3) in pyridine at 22° for thirty minutes, concentrated to dryness, dissolved in ethyl acetate and then analysed by GC-MS. Authentic D-glucose and L-malic acid were treated with racemic and (+)-2-butanol in the same way and injected as references.

Results and discussion

The compound giving green colour with anisaldehyde-sulphuric acid was isolated from S. pereskiifolium as described in the experimental section. Analysis of the ¹H and ¹³C-NMR spectra (Table 1 and 2) showed that the substance consisted of one sugar residue and an aglycone which had one CH₂ group, one CH group and two carbonyl carbons. ¹H-NMR chemical shifts and coupling constants of the signal from the sugar residue indicated a D- glucopyranoside. The ¹H- and ¹³C-NMR chemical shifts of the CH-signal indicated the presence of a CH-O group (Table 1). Positive FAB-MS produced an ion at m/z 319 (M+Na⁺) which corresponds to a molecular weight of 296 for the compound. These data, together with the sugar analysis and the determination of the absolute configuration, demonstrated that the substance consists of a b-D-glucopyranosyl group, linked to a hydroxylated dicarboxylic acid. The latter was isolated after acid hydrolysis of the glucoside and separation of the products by chromatography on Bio-Gel P-2.

The ¹H-and ¹³C-NMR spectra of the dicarboxylic acid were compared and found to be identical with spectra of malic acid.

Determination of the absolute configuration of the acid by GC-MS after reaction with 2-(+)-butanol demonstrated it to be L-malic acid. No separation of the D-and L- forms of malic acid could be obtained after the hydroxyl group of the butyl ester was silylated. On the basis of these results, structure 1(2-O-b-D-glucopyranosyl-l-malic acid) was proposed for the isolated compound. This compound inhibited electrically stimulated contractions of the Guinea pig ileum eight times more than the original total aqueous extract. To our knowledge this compound has never been found in higher plants, but itself and the similar D-tartaric acid glucoside have been synthesized by Helferich and Arndt (1965).

Acknowledgements

This work-was partly sponsored by the International Program in the Chemical Sciences at the University of Uppsala, Sweden, which is hereby gratefully acknowledged. Preliminary work was done at the Department of Traditional Medicines and Drugs Research Centre, Kenya Medical Research Institute. A traditional doctor, Mr. C. Obuya, is also gratefully acknowledged for the basic information he gave on the use of the plant.

Table 1: ¹H-NMR. Chemical shifts (d values) of 1 isolated from Synadenium pereskiifolium and of L-malic acid (coupling constants Hz in parentheses)

Compound	H-1	H-2	H-3	H-4	H-5	H-6a	H-6b	H-2	H-3a	H-3b
Glucoside (H+)	4.58 (7.9)	3.34 (9.1)	3.50 (9.2)	3.41 (9)2	3.43	3.88 (1.6,5.1)	3.72 (12.3)	4.78 (5.7)	2.991	2.991
Glucoside (Na+)	4.47 (7.9)	3.36 (9.2)	3.50 (9.2)	3.40 (9)2	3.41	3.89 (1.8,5.5)	3.70 (12.5)	4.55 (9.5,3.3)	2.66	2.49 (-14.7)
L-malic acid (H+)								4.61 (6.8,5.0)	2.93	2.84 (-16.5)
L-malic acid (Na+)								4.28 (9.3,3.5)	2.67	2.40 (-15.6)

Notes

1. The coupling constant could not be obtained from the spectrum. 9 Hz gave the best result in spin simulation experiments.

2. Unresolved signals.

Table 2: 13C-NMR Chemical Shifts (d values) of 1 isolated from Synadenium pereskiifolium and L-malic acid

Compound	C-1	C-2	C-3	C-4	C-5	C-6	C-1	C-2	C-3	C-4
Glucoside (H+)	102.87	73.97	76.55	70.41	76.87	61.63	174.92	74.47	38.62	174.54
Glucoside (Na+)	102.50	74.14	76.99	70.58	76.98	61.81	179.98	79.12	42.92	179.60
L-malic acid (H+)							176.60	67.07	38.98	174.62
L-malic acid (Na+)							177.71	63.53	39.65	176.66

References

Adjanohoun. (ed.) (1983): Traditional Medicine and pharmacopoeia. Agence de Cooperation Culturelle et Technique (Agency for Cultural and Technical cooperation), Paris.

Gerwig, G.J., Kamerling, J. P. and Vliegenthart, J.F.G. (1978): Carbohydr. Res. 62: 349.

Helferich, B. and Arndt, O. (1965): Ann. Chem. 686: 206.

Kokwaro, J. O. (1976): Medicinal Plants of East Africa. East African Literature Bureau. Nairobi.

Nodharni, A. K. (1976): Dr. K. M. Nadkarni's Indian Materia Medica. Popular Prakashan Private Ltd. Bombay.

Oliver, B. (1960): Medicinal Plants of Nigeria, Nigerian College of Arts, Science and Technology, Ibadan.

Samuelsson, G., Kyerematen, G. and Farah, M.H. (1985): J. Ethnopharmacol. 14: 193.

Watt, H.M. and Breyer-Brandwijk, M.G. (1962): The Medicinal and poisonous plants of Southern and Eastern Africa. E. & S Livingstone Ltd. London: 437.

Abietane diterpene quinones from lepechinia bullata

L. T. JONATHAN

Faculty of Science, Chemistry Department
National University of Lesotho
P.O. Roma 180
LESOTHO

ABSTRACT

Three cytotoxic abietane diterpene quinones, horminone, 7-O-methylhorminone and 6,7-dehydroroyleanone have been isolated for the first time from a methanol (MeOH) extract of Lepechinia bullata (Kunth) Epling (Labiatae). 7-O-methylhorminone is a new natural product, whose structure was unambiguously determined through ¹H-¹³C long range homonuclear correlation (COSY) and heteronuclear correlation (HECTOR) experiments. To date, only a few diterpene quinones have been found to display antitumor activity. In the present study, the three isolates were found to inhibit the growth of P-388 cells although horminone and 7-O-methylhorminone were only marginally active, according to the guidelines of the National Cancer Institute. The compounds did not however, exhibit any significant cytotoxity against KB cells. They represent the first examples of diterpene quinones of the royleanone type to be found cytotoxic against mammalian tumor cells, although horminone has previously been reported to inhibit the growth of Trypanosoma cruzi.

Introduction

Lepechinia bullata (Kunth) Epling (Labiatae), a medicinal plant growing in Colombia, South America, was investigated for antitumour activity. A methanol (MeOH) extract of the above ground parts of the plant was found to be active against P-388 (murine leukaemia) cells (ED₅₀=14.5 mg/ml), but far less sensitive in KB (nasopharyngeal carcinoma) cells (ED₅₀ 40.5 mg/ml).

Phytochemical screening of the bioactive MeOH extract afforded three cytotoxic diterpene quinones, viz., horminone (Fester et al., 1956), 7-O-Methylhorminone (Montes, 1969) and 6,7- dehydroroyleanone (Alpandes et al., 1972). 7-O-methylhorminone is a new natural product whose spectroscopic properties were very similar to those of horminone, thus justifying the royleanone type structure (See also Silver, 1968; Delgado et al., 1986):

Lepechinia bullata has not previously been investigated. Other Lepechinia species such as Lepechinia chalepensia (Fester et al., 1956), Lepechinia floribunda (Montes, 1969), Lepechinia speciosa (Alpandes et al, 1972), Lepechinia salviae (Montes et al., 1983), and Lepechinia graveolens (Riscale and Retamar, 1973) have been analysed for their essential oil content. Diterpenes and triterpenes have also been isolated from Lepechinia chamaedryoides (Silva, 1968) and Lepechinia glomerata (Delgado et al., 1986). The isolation and biological screening of the three abietane diterpene quinones from Lepechinia bullata is reported in this paper.

Figure

Materials and methods

Plant material

The aerial parts of Lepechinia bullata were collected in Colombia in May 1976, by a USDA team. Voucher specimens have been deposited at the National Herbarium, Washington D.C., U.S.A.

Isolation and identification

The crude methanol extract, after being washed with petroleum ether, was partitioned between chloroform (CHCl₃) and aqueous MeOH. The CHCl₃ fraction was chromatographed over silica gel, using CHCl₃ as eluting agent. Fractions (500ml) were collected and combined on the basis of thin layer chromatography (tlc) analysis (see fractionation scheme). Fractions 6-19, on standing in the cold room overnight, deposited an orange precipitate, which was purified by preparative tic and recrystallised from CHCl₃ to give fraction 3 (see Fig. 1). Fractions 47-48, when left overnight in the cold room, deposited a light-green substance which, on repeated chromatography and recrystallisation, gave Fraction 1. Complete spectral analysis (UV, IR, MS ¹H- and ¹³C-NMR) of Fractions 1 and 3, gave data which were in close agreement with those previously reported for horminone (Hensch et al., 1975) and 6,7-dehydroroyleanone (Hensch et al., 1975), respectively.

Flash chromatography, followed by preparative tic of the yellowish- brown solid obtained by evaporation of fractions 31-42, gave the new compound 2 as yellow needles, mp 126-128°C. It was assigned the structure 7-O-methylhorminone, based on its spectroscopic properties. Its mass spectrum displayed a molecular ion at m/z 346, 14 amu higher than that of horminone (m/z 332), shown by the presence of the methoxyl signal, in both the ¹H- and ¹³C-NMR spectra (d3.45 and 57.3 ppm, respectively). The compound was, therefore, most likely a derivative of horminone, with a methoxyl group at either C-7 or C-12. 12-O-methylhorminone has been synthesised and characterised by Hensch et al. (1975). The 7-O-methyl analogue is hitherto unknown.

Further comparison of the ¹H and ¹³C-NMR spectra of 1 and 2 (Table 2 and 3) showed that the most significant difference between them lies in the chemical shift values for H7 and C7. The methoxyl group in 2 caused the H7 signal to move upfield to d4.32, from d4.73 in 1, and concurrently, C7 absorbed downfield at d0.8 from d63.2. The results are consistent with a methoxyl substituent at C7 and not C12 of horminone.

The uv spectra of 2 provides further evidence for a methoxyl substituent at C7. The absorption at 411 nm exhibited a significant bathochromic shift to 524 nm on addition of NaOH, indicating the presence of a quinonoid hydroxyl group at C12. Similar uv shifts have been reported in diterpene quinones bearing a quinonoid hydroxyl function (Lin et al, 1989).

The stereochemistry at C7 was determined from the ¹H-NMR spectrum. Kupchan et al. (1968, 1969) have compared the H-7bH signal of horminone with the H-7ah of taxoquinone, its 7-epimer. They found that at 60 MHz, the H-7bH appeared as a multiplet with W1/2 = 20 Hz, whereas the H-7bH signal was a broad singlet, with W1/2 == 8Hz. In our work, the H7 of 2, measured at 300MHz, was observed as a doublet of doublets with J = 2 and 4Hz, consistent with a b orientation of H7. Compound 2 was, therefore assigned the structure 7-O-methylhorminone.

Biological screening

The MeOH extract and the three isolates were tested for antitumour activity in KB and P-388 cell cultures, according to standard procedures as described previously (Pezzuto et al., 1983, and Arisawa et al, 1984). The results are shown in Table 1. All isolates inhibited the growth of P-388 cells, although 1 and 2 were only marginally active, according to the guidelines of the National Cancer Institute (Geran et al., 1972). They did not, however, show any significant cytotoxicity against KB cells. The three compounds represent the first examples of diterpene quinones of the royleanone type, to be found cytotoxic against mammalian tumour cells. It is worth noting that in both KB and P-388 systems, the unsaturated 6,7-dehydro compound (3) is more active than the 6, 7- saturated structures, leading to speculation that the antitumour activity of these compounds depends on the substitution pattern at the C6-C7 position of these molecules.

Acknowledgements

This work was supported by the Fulbright Program of the United States of America. The Program for Collaborative Research in the Pharmaceutical Sciences (PCRPS), College of Pharmacy, University of Illinois at Chicago, is gratefully acknowledged, for providing the facilities for this investigation. My special thanks go to Dr. Chun-Tao Che, Prof. Harry H.S. Fong, and Prof. Norman R. Farnsworth.

Table 1. Data obtained from pharmacological testing of KB cells (nasopharyngeal carcinoma) and P-388 cells (murine leukemia) with Lepechinia bullata plant extracts and pure compounds

Compounds		ED50
	KB cells	P-388 cells
Crude MeOH extract	40.5 mg/ml	15.5 mg/ml
Horminone	20.2 mg/ml	4.6 mg/ml
7-O-methylhorminone	13.0 mg/ml	4.8 mg/ml
6,7-dehydroyleanone	5.7 mg/ml	1.6 mg/ml

Table 2. Summary of 1H-NMR of Extracts 1 and 2 (300 MHz, CDCI₃)

	d (ppm)
Proton	Horminone	7-O-methylhormine
H - 7b	4.73 (d)	4.31 (dd)
H - 15	3.16 (septet)	3.18 (septet)
H - 1b	2.16 (ddd)	2.68 (ddd)
H - 6a	1.96 (d)	2.04 (d)
H - 2b	1.72 (m)	1.70 (m)
H - 5	1.55 (hidden)	1.57 (hidden)
H - 3a,b	1.5 - 2.7 (m)	1.4 - 1.6 (m)
H - 6a	1.4 - 1.5 (m)	1.35 (ddd)
H - 2	1.2-1.3 (hidden)	1.2- 1.3 (m)
Me - 16	1.21 (d)	1.19 (d)
Me - 17	1.22 (d)	1.22 (d)
Me - 20	1.22 (s)	1.22 (d)
H - 1a	1.1 - 1.2 (m)	1.1- 1.2 (m)
Me - 1d	0.98 (s)	0.95 (s)
Me - 19	0.90 (s)	0.91 (s)
7 - Ome	-	3.45 (s)

Table 3. Summary of data of C-13-NMR of Compunds 1 and 2 (90.8 MHz, CDC1₃)

	d(ppm)
Carbon	Horminone (1)	7-O-methyl-horminone (2)
C - 14	189.0	186.4
C - 11	183.8	184.1
C - 12	151.1	150.6
C - 9	147.8	147.8
C - 8	143.1	141.4
C - 13	124.1	124.7
C - 7	63.2	70.7
C - 5	45.7	45.5
C - 3	41.0	41.0
C - 4	39.1	39.2
C - 1	35.7	35.7
C - 18	33.1	33.0
C - 10	33.0	33.0
C - 6	25.7	22.1
C - 15	23.9	24.2
C - 19	21.7	21.9
C - 17	19.8	19.9
C - 16	19.7	19.7
C - 2	18.8	18.8
C - 20	18.3	18.5
C - Ome	-	57.3

Figure 1: Fractionation scheme for the extracts from Lepechinia bullata

Antimicrobial activity of Tanzanian traditional medicinal plants

M.R. KHAN and M.H.H. NKUNYA

Department of Chemistry, University of Dar es Salaam
P.O. Box 35061, Dar es Salaam, Tanzania

ABSTRACT

A large number of plants used in traditional medicine were screened for antimicrobial activity. In the preliminary screening, Staphylococcus aureus (gram positive bacteria) and Escherichia coli (gram negative bacteria) were used to differentiate between active and non-active plant extracts. The extracts which showed activity were then screened for their antigonococcal and also for antifungal activity. A number of active plants were then phytochemically investigated to isolate the active components. A large number of different types of non-active compounds were also isolated and identified. There is some correlation between the activities and the traditional medicinal uses of the plants studied. Some of the compounds isolated could be responsible for the activity and use of the plants. This paper gives only the in-vitro screening and the results should be used with caution when applied to in-vivo effectiveness in humans. Screening needs to be done in-vivo and the toxicity aspect has to be studied very thoroughly before such crude plant extracts could be given as safe treatment with no serious consequences.

Introduction

In African and most developing countries traditional medicine still forms the backbone of rural medical practice. Medicinal herbs are extensively used for various ailments in these countries. This indicates that some of these medicines, if scientifically evaluated and standardized, could make very valuable medicaments. However, although a number of American (Lucas et al., 1951) and Australian (Atkinson et al., 1955) medicinal herbs have been screened for their medicinal properties, up to now there seem to be no serious attempts to evaluate African medicinal plants in a collective form for their biological activities and medicinal usefulness. However, there are scattered reports of such evaluations for individual or small groups of plants, as it will be noted in various presentations in this conference.

In the literature, it can be noted that Nickell (1959) is among the first researchers to compile an extensive review on biological (antibacterial) activity of vascular plants. Nickell's list of plants included only a few of Tanzanian medicinal plants. We therefore considered it worthwhile to investigate the in vitro antibacterial and antifungal activities of some of the Tanzanian medicinal plants, and ultimately to isolate and identify the active constituents (Sawhney et al., 1978a; Sawhney et al., 1978b: Khan et al., 1979).

We chose to screen the medicinal plants for antifungal activity because, of all human microbial infections, fungal diseases are the most difficult to modify in their course, or to prevent (Lucas et al., 1973; Taylor et al., 1961). It is now becoming more evident that the incidence of such diseases is increasingly becoming prominent.

From the literature (Kokwaro, 1976; Watt et al., 1962) and personal communications with Tanzanian traditional medical practitioners, we established that a number of herbs are used for the treatment of skin diseases, and many of them are said to be very effective. Thus the fruits of Solanum incanum, a weed which is widely distributed in East Africa, are extensively used for the treatment of cutaneous mycotic infections and other pathological conditions. The therapeutic action of the fruits has been attributed to solanine and related glycoalkaloids (Beaman-Mbaya et al., 1976). Similarly, the juice of Emilia sagittata is used for ring worms and athletic's foot. Although no chemical work is reported on this plant, a very potent antimicrobial and pharmacological agent, emiline (1), has been obtained from another plant of the same genus, E. flammea (Tomczyk et al., 1971).

Apart from using Staphylococcus aureus and Escherichia coli as test bacteria, we also included the essay of the crude plant extracts for their antigonococcal activity. This is because gonorrhoea is among the most common venereal diseases, both in rural and urban populations in Africa (Becker, 1973). Despite the introduction of sulphonamides and antibiotics, a large proportion of rural populations in developing countries still rely on local herbs for the treatment of gonorrhoea. Thus, in West Africa for example, cottonwood tree (Bombax sp.), Alchronea cordifolia, A. floribunda, Mussaenda elegans, Craterspermum laurinum and Aframomum baumannii are commonly used (Harley, 1970). There are also similar example in East Africa (Kokwaro, 1976).

In this paper we will give an overview of the results on the screening of crude plant extracts for their antibacterial, antigonococcal and antifungal activity and the phyto-chemical investigations on some of the most active plants.

Antibacterial activity

In all, 134 plant extracts were tested for their activity against S. aureus and E. coli in vitro. An extract which failed to inhibit the growth of the test bacteria was regarded as being inactive. Results are summarized in Table 1, in which the inactive extracts are not shown.

Phytochemical investigations on some of the most active extracts have revealed the active constituents of the plants. Thus the activity of Euclea natalensis can be attributed to 7- methyljuglone (2), mamegakinone (3) and diospyrin (4). These compounds, which were isolated from the plant, have been found to be active against S. aureus and a few other bacteria (Table 2).

The antibacterial activity of Harrisonia abyssinica root bark, which showed an activity against S. aureus, comparable to 5 units of penicillin G, has been traced to be due to the limonoid harrisonin (5) (Kubo et al., 1976). The latter compound, which was the only active component of this plant, showed a minimum inhibitory concentration of 5 mg/ml (Mosile, 1980).

Another most active plant is Acacia nilotica. This plant is known to contain phenylethyl alkaloids and flavonoids. Although these compounds have not been tested, we found the activity to be concentrated in the acidic fraction of the extract, which contains the flavonoids.

Active compounds which have been isolated from some of the most active plant extracts are shown in Chart 1.

Antigonococcal activity

In this category of assay, extracts from 88 Tanzanian medicinal plants were tested for their in vitro activity against Neisseria gonorrhoea isolates from clinical cases, which were isolated and maintained at the Department of Microbiology and Immunology, Faculty of Medicine, University of Dar es Salaam (Sawhney et al, 1978a). Results are shown in Table 3. It is interesting to note that some of the plants used locally for the treatment of gonorrhoea are very active against the pathogenic bacteria. Furthermore, 82% of the plants listed in Table 4 were also active against S. aureus. More than 40% of the plant extracts without antigonococcal activity showed various levels of inhibition of S. aureus. This, in a way, ruled out the effect of nonspecific factors, such as acidity, on the observed activity.

Antifungal activity

In all, 124 plants were screened for activity against the common dermatophyte, Trichophyton mentagrophytes, as well as Candida albicans. Results are summarized in Table 4.

As it can be noted in Table 4, the highest level of antifungal activity was exhibited by extracts of Emillia sagittata, Securrinega virosa (pulp) and Sida serratifolia (roots) (Sawhney et al., 1978b). Apparently, none of these plants is used to treat dermatomycoses in East Africa. Instead these plants are used for miscellaneous ailments, such as eye inflammation, topical dressing for wounds and contusions, diarrhoea, gonorrhoea, pneumonia, pulmonary tuberculosis and dysentery, most of which are bacterial diseases (Kokwaro, 1976; Watt et al, 1962). Incidentally, among the above plants only S. serratifolia showed antibacterial activity in vitro (Table 1). Such results may suggest that either the antibacterial activity is exhibited only in vivo, in patients, or the plants are used just as a matter of tradition. Again, the observed antifungal activity, despite the plants not being used traditionally for fungal related diseases, gives us a good indication that a lot is yet to be discovered regarding the diverse usefulness of medicinal plants.

Phytochemical investigations

We have carried out extensive phytochemical investigations on some of the most active plants shown in Tables 1, 4 and 5, with the aim of isolating the active constituents. Thus from Euclea natalensis we isolated several naphthaquinones, among which the active ones are listed in Table 2 (Khan et al., 1979).

Several triterpenoids and naphthaquinones have been isolated from various Diospyros species (Ebenaceae), but only 7-methyljuglone, diospyrin and mamegakenone were the active compounds in this series. Eleven Cassia species have been analysed for their constituents, and in addition to emodine (6), aloe-emodine (7) and barakol (8), several other anthraquinones have been isolated, some of which were obtained for the first time (Mutasa, et al., 1990).

Maerua angolensis (Capparidaceae) is among the plants which exhibited a high antifungal activity. We have isolated several C₁₂, C₁₄ and C₁₈ fatty acids and esters from this plant, and most of these compounds showed antifungal activity (Nkunya, 1985).

Among the plants of the family Annonaceae, which were included in the screening tests, were those belonging to the genus Uvaria. In the literature some of these plants are reported to possess a wide range of biological activities. Furthermore, these plants have been found to contain compounds with interesting chemical structures, some of which are also the active components of the plant extracts. These findings prompted us to carry out extensive phytochemical investigations of these plants. In the course of these investigations, we have isolated more than forty compounds from nine Uvaria species found in Tanzania. An account of these compounds, regarding their biological activities, has been given by Nkunya (1990, this conference), in a paper on the antimalarial activity of the compounds. Apart from this, the compounds have shown activity against some bacteria and tumour cells. Among the active compounds are (+)-b-senepoxide (9), (+)-pandoxide (10) and (-)-pipoxide (11) (Nkunya et al, 1986). Results on the antibacterial activity are shown in Table 5.

Conclusion

The results discussed in this paper do not claim that the plants we have investigated and the pure compounds therefrom are safe medicines. Their efficacy and safety can only be established by very careful toxicity and pharmacological studies, followed by clinical trials using usual protocols. Our results definitely have provided a basis for further investigations on similar lines, as well as on the toxicity and pharmacological aspects of the extracts, and pure compounds. We hope that the leads presented here will be pursued exhaustively by the scientific community.

References

Atkison, A. and Brice, C. (1955). Austr. J. Exptl. Biol. Med. Sci. 33: 547 - 554.

Beaman-Mbaya, V. and Muhammed, S. I. (1976). Antimicrob. Agents Chemother. 9: 920 - 924.

Becker, N. L. (1973). In Clinical Medicine in Africans in Southern Africa. Campbell, G.D., Seedat, Y.K. and Daynes, G. (Eds). Churchill/Livingstone, London: 465.

Harley, G.W. (1970). Native African Medicine, Frank Cass, London.

Khan, M.R., Mutasa, S.L., Ndaalio, G., Wevers, H. and Sawhney, A.N. (1978): Pakistan J. Sci. Ind. Res. 21: 197 - 199.

Khan, M.R., Ndaalio, G., Nkunya, M.H.H., Wevers, H. and Sawhney, A.N. (1980). Planta Med., Suppl.: 91 - 97.

Kokwaro, J. O. (1976). Medicinal Plants of East Africa, East African Literature Bureau, Nairobi.

Kubo, I., Tanis, S. P., Lee, Y., Miuva, F., Nakanishi, K. and Chapya, A. (1976). Heterocycles 5: 485.

Lucas, A. O. and Gilles, H.M. (1973). A short Textbook of Preventive Medicine for the Tropics. English University Press, London: 127.

Lucas, E. H., Lickfield, A., Gottshall, F. and Jennings, J. C. (1951). Bull. Torrey Bot. Club 78: 310 - 321.

Mosile, F. W. (1980). Chemical studies and antimicrobial activity of some Tanzanian medicinal plants: M.Sc, Thesis, University of Dar es Salaam.

Mutasa, S.L., Khan, M.R. and Jewers, K. (1990). Planta Med. 56: 244.

Nickell, L. G. (1959). Econ. Bot. 13: 281 - 318.

Nkunya, M. H. H. (1985). A search for potentially useful compounds from some Tanzanian plants: In Proc. Sci. Symp. Univ. Dar es Salaam, Publisher: Tanzania Commission for Science and Technology: 73-75.

Nkunya, M. H. H. and Weenen, H. (1986). Chemical investigations of a Tanzanian medicinal plant: Uvaria pandensis Verdc (Annonacese). In: Proc. 3rd Internat. Chem. Conf. Africa, Lome (Togo): 313 - 317.

Nkunya, M. H. H. (1990). Chemical evaluation of Tanzania Medicinal Plants for active constituents as a basis for the medicinal usefulness of the plants. In Proc., this conference.

Sawhney, A. N., Khan, M.R., Ndaalio, G., Nkunya, M.H.H. and Wevers, H. (1978a). Pakistan J. Sci. Ind. Res. 21: 189 - 192.

Sawhney, A. N., Khan, M.R., Ndaalio, G., Nkunya, M. H. H. and Wevers, H. (1978b). Pakistan J. Sci. Ind. Res. 81: 193 - 196.

Taylor, E. P. and D'Arcy, P. F. (1961). Progress in Medicinal Chemistry, Plenum Press, New York: 220.

Tomaczyk, H. and Kohlmuenzer, S. (1971). Herba Pol. 17, 226 (Chem. Abstr. 1972, 77: 1984)

Watt, J. M. and Breyer - Brandwijk, M. G. (1962). Medicinal and Poisonous Plants of Southern and Eastern Africa, 2nd Ed., Livingstone, London.

Table 1: Susceptibility of Staphylococcus aureus and Escherichia coli to various plant extracts

Antibacterial activity
Name of the plant	Family	Part	Traditional uses	Staphylococcus aureus	Escherichia coli
Anona senegalensis Pers.	Annonaceae	Bark	Intestinal worms, guinea worms, dysentery	+	0
Uvaria acuminata Oliv.	Annonaceae	Roots	Epilepsy, sunstroke, tonsillitis, lunasy	+	0
Uvaria acuminata Oliv.	Annonaceae	Leaves	Epilepsy	+	0
Dictyophleba lucida	Apocynaceae	leaves	No known use	++	++
Pierre Plumeria rubra L.	Apocynaceae	Bark	Itching, diarrhoea gonorrhoea, dropsy, purgative, skin diseases, syphilis	++	0
Kigelia africana (Lam.).) Berth	Bignoniaceae	Bark	Wounds, sores, gynaecological conditions, ulcers, abscesses, dysentery	++	0
Tecomaria capensiss Spach.	Bignoniaceae	Leaves	Pneumonia, bleeding gums, diarrhoea, enteritis	+++	++
Ehretia amoena Klotzch	Boraginaceae	Root-bark	For pains about the waist (stitch)	++	+
Boscia salicifolia O.	Capparidaceae	Bark	Chiufa, various women's diseases	++	0
Boscia salicifolia O.	Capparidaceae	Leaves	Chiufa, remedy for fever in cattle	++	+
Maerus angolensis D.C.	Capparidaceae	Bark	Roots used for homocidal purposes, treatment of lupus, influenza, toothache	+ +	0 0
Carica papaya L.	Caricaceae	Roots	Venereal diseases, anti-helmintic, akin	0	+
Elaeodendron schlechteranum (L.)	Celastraceae	Roots	Elaeodendron sp. to abscesses and carbuncles	++	0
Vernonia hildebrandtii V.	Compositae	Leaves stem	Cough, strangulated hernia, stomach troubles	+++	0
Cyperus rotundus L.	Cyperaceae	Tuber	Diuretic, emmenagogue, liver and heart desease remedy, headache cure, carminative	+++	0
Tetracera boiviniana B.	Dilleniceae	Root-bark	No known use	++	0
Diospyros mespiliformis Hoechst ex DC	Ebenaceae	Leaves	Anthelmintic, wounds & sores, leprosy, dysentery, coughs	+	0
Euclea natalensis A.DC.	Ebenaceae	Root-bark	Gonorrhoea, syphilis, hookworm, relief of toothache, ulcers	++	0
Acalypha fruticosa F.	Euphorbiaceae	Leaves	Cholera, stomach ache coughs, chest pains	++	0
Euphorbia hirta L.	Euphorbiaceae	Plant	Gonorrhoea, dysentery, boils, coughs, ophtholmic, wounds.	+++	++
Phyllanthus niruri L.	Euphorbiaceae	Plant	Gonorrhoea, ulcers jaundice, sores urino-genital diseases.	++	++
Phyllanthus reticulatus P.	Euphorbiaceae	Leaves	Gonorrhoea, venereal sores, hookworms, anaemia.	++	0
Pseudolachmaestylis maprouneaefolia Pax	Euphorbiaceae	Bark	Stomachache, cathartic	++	0
Ricinus communis L.	Euphorbiaceae	Plant	Venereal diseases, ulcers diarrhoea, fungicidal, eardrop	++	0
Seccurinega virosa B.	Euphorbiaceae	Roots	Gonorrhoea	+	++
Hoslundia opposita Vahl	Labiatae	Plant	Gonorrhoea, cystitis, coughs, wounds, liver disease, blennorrhoea, hookworms.	+++	0
Cassytha filiformis L.	Lauraceae	Plant	For vermin, gonorrhoea dysentery, syphilis, snake bite wounds	++	0
Acacia mellifera Vahl	Leguminosae	Bark	Syphilis, pneumonia, malaria, sterility, stomachache	+	0
Acacia nilotica Del.	Leguminosae	Plant	Tuberculosis, pneumonia, gonorrhoea, diarrhoea, smallpox	+++	++
Acacia robusta Burch.	Leguminosae	Root-bark	No known use	++++	++
Acacia sieberiana DC.	Leguminosae	Bark	Gonorrhoea, stomachache, diarrhoea, haemorrhage.	+++	0
Bauhinia reticulata DC.	Leguminosae	Plant	Dysentery, leprosy, roundworms, anthrax, malaria, cough	++	0
Caesalpinia pulcherrimai Swartz	Leguminosae	Flowers	Lung disease, fever, skin diseases	+	0
Caesalpinia pulcherrima Swartz	Leguminosae	Bark	Lung disease, fever skin disease	++	0
Caesalpinia pulcherrima Swartz	Leguminosae	Root-bark	Lung disease, fever skin diseases	++	0
Cassia abbreviata Oliv.	Leguminosae	Dry - roots	Gonorrhoea, syphilis, diarrhoea, dysentery pneumonia, malaria	++	0
Cassia fistala L.	Leguminosae	Bark	Dysentery, blackwater fever, anthrax, malaria	+++	0
Cassia obtusifolia L.	Leguminosae	Whole plant	Stomach troubles	++	+
Dichrostachys cinerea W.	Leguminosae	Stem & branches	Gonorrhoea, syphilis, skin diseases	+++	+
Lonchocarpus bussei Harms.	Leguminosae	Bark	Gonorrhoea, cough	++	0
Peltophorum petocarpum (DC.) K.	Leguminosae	Bark	Dysentery, diarrhoea, colic, sore eyes	++	0
Pongania pinnata (L.)P.	Leguminosae	Leaves Root-bark	Scabies, cutaneous infection	++	0
Pongania. Pinnata (L.)P.	Leguminosae	Seeds	Scabies, cutaneous infection	++	0
Asparagus falcatus L.	Liliaceae	Leaves	Syphilis	++	0
Sida serratifolia L.	Malvaceae	Leaves	Gonorrhoea	+++	0
Sida serratifolia L.	Malvaceae	Roots	Gonorrhoea	+++	0
Psidium guajava L.	Myrtaceae	Leaves	Diarrhoea, skin diseases	++	0
Brackenridgea zanguebarica Oliv.	Ochnaceae	Root-bark	Wounds, snakebites	+	0
Ziziphus pubescens Oliv.	Rhamnaceae	Leaves	Pneumonia, diarrhoea dysentery, wounds, skin diseases	+++	0
Lamprothamnus zanguebaricus Hiern.	Rubiaceae	Leaves	No known use	++	0
Fagara chalybaea Engl.	Rutaceae	Root-bark	Diarrhoea, coughs, malaria, toothache	++	0
Allophylus rubifolius (A.Rich.)	Sapindaceae	Roots	Diarrhoea, toothache	+	0
Solanum incanum L.	Solanaceae	Plant	Pneumonia, ringworms, liver disease, gonorrhoea, syphilis, ear ache	+	0
Solanum incanum L.	Solanaceae	Fruits	Dandruff, skin diseases, sores and wounds	++	0
Harrisonia abyssinica Oliv.	Simaroubaceae	Root-bark & twig	Skin diseases, haemorrhoids	++++	0
Grewia forbesii Harv. ex Mast.	Tiliaceae	Bark & roots	Rheumatism, lumbago, stiff neck	+++	0
Lantana camara L.	Verbenaceae	Leaves	Coughs, sore throat, colds, conjunctivitis, toothache	+	0
Premna chrysoclada G.	Verbenaceae	Leaves	Ulcers, venereal diseases	++	0
Vitex fischeri. G	Verbenaceae	Leaves	Chronic venereal diseases, epilepsy as sedative, skin diseases.	++	0
Rhoicissus rovoilii P.	Vitaceae	Roots	Wounds, optholmic remedy	+	0

Table 1a: Sensitivity of test organisms against a number of standard antibiotics

Standard Antibiotics	diameter of Zones of inhibition (mm)
	+ 10-15	++ 15-20	+++ 20-25	++++ above 25	Test Organisms
Penicillin G. (Units)	2	3	4	5
Septrin (SXT) (mg)	15	20	25	30
Tetracycline (mg)	25	32	42	60	Staphylococcus aureus (Oxford)
Streptomycin (mg)	7	9	12	15
Sulphathiamoxazole (mg)	12	18	24	30
Nalidixic acid (mg)	15	24	30	35
Furadantoin (mg)	75	100	125	130	Escherichia coli (055)
Gentamycin (mg)	23	30	36	43

Table 2: Susceptibility of some microorganisms to some naphthoquinones

Bacteria	Zones of inhibition (mm)
	7-Methyl-juglone	Diospyrin	Mamegakinone
Klobsiella aeroganesae (from urine)	11	9	11
Shigella dysenteriae	14	14	9
Shigella flexnerii	12	11	0
Corynebacterium diphtheriae	13	14	-
Bacillus anthracis	17	13	-
Bacillus cereus	9	10	0
Salmonella hidelberg	8	8	8
Hamophilus influenzae	11	12	10
Pseudomonas aureginosae	0	0	0
Escherichia coli	0	0	0
Clostridium wolchii	8	0	0
Staphylococcus aureus	11	0	22
Neisseria gonorrhoeae	24	0	14

The 10 - 15mm zone of inhibition is comparable to the one caused by 25 mg of tetracycline

Table 3: In vitro antigonococcal activity of some Tanzanian medicinal plants

Plant	Family	Part	Traditional uses	Antigonococcal activity
Sclerocarya caffra Sond.	Anacardiaceae	Bark	Dysentery, diarrhoea, gangrenus, rectitis, insecticide	+
Uvaria acuminata Oliv.	Annonaceae	Leaves	Epilepsy	++
Kigelia africana (Lam.) Benth.	Bignoniaceae	Bark	Wounds, sores, for gynaecological conditions, ulcers, abscesses, dysentery	++
Tecomaria capensis Spach.	Bignoniaceae	Leaves	Pneumonia, bleeding gums, diarrhoea, enteritis	++
Tetracera boiviniana Baill.	Dileniceae	Roots	No known use	+
Euclea natalensis A.DC.	Ebanaceae	Root-bark	Gonorrhoea, diarrhoea, dysentery, bleeding gums	+
Phyllanthus reticulatus P.	Euphorbiaceae	Leaves	Gonorrhoea, venereal sores, hookworms, anaemia	++
Ricinus communis L.	Euphorbiaceae	Plant	Venereal diseases, ulcers, diarrhoea, fungicidal, eardrop	+++
Acacia nilotica Del.	Leguminosae	Bark	Tuberculosis, pneumonia, gonorrhoea, diarrhoea, smallpox	++++
Albezia harveyi Fcurn	Leguminosae	Roots	Any intestinal troubles	+
Bauhinia reticulatus DC.	Leguminosae	Plant	Dysentery, leprosy, roundworms, anthrax, malaria, cough	+
Caesalpinia pulcherrima Swartz.	Leguminosae	Flowers	Lung diseases, fever, skin disease	+
Cassia abbreviata Oliv.	Leguminosae	Dry roots	Gonorrhoea, syphilis diarrhoea, dysentery pneumonia, malaria	+
Cassia obtusifolia L.	Leguminosae	Whole plant	Stomach troubles	+++
Lonchocarpus bussei Harms.	Leguminosae	Leaves, roots & bark	Gonorrhoea, cough	+
Malvastrum coramandelianum (L) Garcke.	Malvaceae	Plant	Wounds, diaphoretic, sores	+
Sida serratifolia L.	Malvaceae	leaves	Pulmonary tuberculosis, diarrhoea	+++
Sida serratifolia L.	Malvaceae	Roots	Gonorrhoea	++
Psidium guajava L.	Myrtaceae	Leaves	Diarrhoea, skin diseases	+
Ziziphus pubescens Oliv.	Rhamnaceae	Stern	Measles, gonorrhoea	+
Fagara chalybaea Engl.	Rutaceae	Root-bark	Diarrhoea, coughs, malaria, toothache	+++
Harrisonia abyssinica, O.	Simarubaceae	Twig & rootbark	Skin diseases, haemorrhoids	+++
Premna chrysoclada G.	Verbenaceae	Leaves	Ulcers, venereal diseases.	+

The following plants did not show any antigonococcal activity:

Acanthaceae: Barleria prionitis L. (roots, leaves and bark); Amaranthes aspera L. (plant);

Anacardiaceae: Rhus natalensis Bernh. (leaves), Lannea stuhlmannii Engl. (leaves);

Annonaceae: Anona senegalensis Pers (bark), Uvaria acuminata Oliv. (roots);

Apocynaceae: Calotropis gigantea Ait. f. (leaves), Dictyophleba lucida Pierre. (leaves, trunk), Nerium oleander L. (leaves), Plumeria rubra L. (bark);

Araceae: Stylochiton hennigii Engl. (roots and leaves);

Boraginaceae: Ehretia amoena Klotzch. (root bark);

Capparidaceae: Boscia salifolia Oliv. (bark, leaves), Maerua angolensis DC. (bark); Carica papaya L. (leaves, roots, (bark);

Celastraceae: Elaeodendron schlechteranum Loes. (roots);

Combretaceae: Combretum zeyheri Sond. (fruits, plant), Terminalia catappa L. (leaves); Compositae: Aspilia natalensis Willd. (roots), Emilia sagittata D.C. (plant);

Convolvulaceae: Bonamia mossambicensis Hall. f. (roots);

Cyperaceae: Cyperus rotundus L. (tuber);

Ebenaceae: Diospyros mespiliformis Hochst ex DC (leaves);

Euphorbiaceae: Acalypha fruticosa Forsk, (roots), Fluggea virosa Baill. (bark), Phylanthus niruri L. (plant), Pseudolachmaestylis maprouncaefolia Pax. (bark), Securinega virosa Baill, (bark, pulp);

Icacinaceae: Pyrenacantha kaurabassana Baill (tuber, green fruits); Labiatae: Hoslundia opposita Vahl. (leaves), Leonotis nepetaefolia R. Br. (plant);

Lauraceae: Cassytha filiformis L. (plant);

Leguminosae: Acacia robusta Burch (rootbark), A. Senegal Wild. (roots), Adenanthera pavonina L. (seeds), Caesalpinia pulcherrina Swartz (bark), Cassia fistula L. (bark), C. amiculata L. (seeds and bark), Desmodium sp. (plant), Dichrostachys cinerea Wight. Am. (roots), Peltophorum petocarpum K. (roots, bark), Pongania pinnata L. (leaves, rootbark, seeds), Pterocarpus angolensis DC (bark), Stylosanthes fruticosa Alston. (plant), Xeroderris stuhlmannii Taub. (plant);

Liliaceae: Asparagus falcatus L. (plant);

Malvaceae: Sida spinosa L. (leaves);

Rhamnaceae: Ziziphus pubescens Oliv (leaves);

Rubiaceae: Lamprathamnus zanguebaricus Hiern. (leaves);

Rutaceae: Citrus aurantifolia Swingle. (roots);

Sapindaceae: Allophylus rubifolius Engl. (stem);

Solanaceae: Withania somnifera Dun (plant);

Sterculiaceae: Dombeya shupangae K. Schum (leaves), Melhania velutina Forsk. (leaves), Waltheria indica L. (flowers, leaves);

Tiliaceae: Corchorus olitorius L. (fruits and seeds), Grewia forbesii Hary ex Mast. (bark and roots), G. Stuhlmannii K. Schum (roots), Trimimfetta rhomboidea Jacq. (bark and roots);

Verbenaceae: Lantania camara L. (leaves), Vitex fischeri Guerke. (leaves), Vitex sp. (roots);

Vitaceae: Cissus integrifolia Manch. (stem), Rhoicissus rovoilii Planch (roots).

Table 4: Susceptibility of fungi to various plant extracts

Plant	Family	Part	Traditional uses	Antifungal activity
Group A
Plumeria rubra L.	Apocynaceae	Bark	Itching, diarrhoea, gonorrhoea, dropsy, purgative, skin disease, warts, syphilis	++
Zizyphus pubescens Oliv.	Rhamnaceae	Leaves	Pneumonia, diarrhoea dysentery, wounds, skin diseases	++
Solanum incanum L.	Solanaceae	Plant	Pneumonia, ringworms, liver disease, gonorrhoea, syphilis, earache	++
Solanum incanum L.	Solanaceae	Fruits	Dandruff, skin diseases, sores, & wounds	++
Harrisonia abyssinica Oliv.	Simaroubaceae	Root-bark & twig	Skin diseases, haemorrhoids.	+++
Waltheria indica L.	Sterculiaceae	Flowers	Skin diseases, syphilis, cleansing wounds, coughs, sores.	+
Vitex fischeri Guerke.	Verbenaceae	Leaves	Chronic venereal disease, epilepsy, as sedative, skin diseases.	+
Group B
Dictyophleba lucida (K. Schum.) Pierre.	Apocynaceae	Leaves	No known use	+++
Dictyophleba lucida (K. Schum.) Pierre.	Apocynaceae	Trunk	No known use	+++
Holarrhena febrifuga Klotzsch.	Apocynaceae	Leaves	Snake bite, venereal diseases, dysentery	++
Ceiba pentandra Gaertn.	Bombacaceae	Leaves	Gonorrhoea and as dressings for wounds	+
Boscia salicifolia Oliv.	Capparidaceae	Bark	Rectal infections	++
Combretum zeyheri Sond.	Combretaceae	Whole plant	Diarrhoea	+++
Emilia sagittata DC.	Composite	Whole plant	For inflammation of eyes, contusion, ulcerative processes, nasal disease syphilis	++++
Bonamia mossammbicensis (Klotzsch.) Hall. f.	Convolvulaceae	Leaves	Wounds	++++
Bonamia messambicensis (Klotzsch.) Hall. f.	Convolvulaceae	Roots	Wounds	+++
Bridelia cathartica B.	Euphorbiaceae	Stem	Purgative, stomach ache	+
Phyllanthus reticulatus P.	Euphorbiaceae	Plant	Gonorrhoea, ulcers, jaundice sores, urogenital diseases	+
Pseudolachnostylis maprouneaefolia Pax.	Euphorbiaceae	Bark	Stomach ache, cathartic	++
Securinega virosa (Wind.) Baill.	Euphorbiaceae	Pulp	Diarrhoea, gonorrhoea, pneumonia	++++
Cassia amiculata L.	Leguminosae	Bark	Headache, toothache	++
Xeroderris stuhlmanii T.-	Leguminosae	Plant	Colds, chest troubles, elephantisis	+
Asparagus falcatus L.	Liliaceae	leaves	Syphilis	+
Hibiscus micranthus L.	Malvaceae	Plant	Earache, bronchitis, renal remedy	++
Sida serratifolia L.	Malvaceae	Leaves	Pulmonary tuberculosis, diarrhoea	+++
Sida serratifolia L.	Malvaceae	Roots	Gonorrhoea	++++
Citrus aurantifolia Swingle.	Rutaceae	Roots	Gonorrhoea, dysentery	++++
Fagara chalybea Engl.	Rutaceae	Root-bark	Diarrhoea, coughs, malaria, toothhache	++
Deinbollia borbonica nica R.	Sapindaceae	Roots	Chest troubles, abdominal pains	+

Plant extracts which did not show any in vitro antifungal activity:

Acanthaceae: Barleria prionitis L. (roots, leaves and bark);

Amaranthaceae: Achyranthes aspera L. (plant);

Anacardiaceae: Rhus natalensis Bernh. (leaves), Lannea stuhlmannii Engl. (leaves);

Annonaceae: Anona senegalensis Pers. (bark), Uvaria acuminata Oliv. (leaves, roots);

Apocynaceae: Calotropis gigantea Ait. F. (leaves), Nerium oleander L. (leaves); Stylochiton hennigii. (roots and leaves);

Bignoniaceae: Kigelia africana Benth. (bark), Tecomaria capensis Spach. (leaves);

Boraginaceae: Ehretia amoena Klotzch. (root bark);

Capparidaceae: Boscia salicifolia Oliv. (leaves), Maerua angolensis DC. (bark, leaves);

Caricaceae: Carica papaya L. (green fruits, bark);

Celastraceae: Elaeodendron schlechteranum Loes. (roots, leaves);

Combretaceae: Combretum zeyheri Sond: (fruits), Terminalia catappa L. (leaves); Compositae: Vernonia hildebranditii Vatke, (leaves and stem), V. cinerea Less. (plant);

Connaraceae: Byrsocarpus orientalis Bak. (plant);

Dilleniceae: Tetracera boiviniana Baill. (rootbark);

Ebenaceae: Diospyros mespiliformis Hochst. ex. DC (leaves);

Euphorbiaceae: Acalypha fruticosa Forsh (leaves, roots), Antidesma venosum E. May. (root bark), Bridelia cathartica Bertol. f. (leaves), Euphorbia hirta L. (plant), Fluggea virosa Baill, (bark), Phyllanthus reticulatus Poir. (leaves), Securinega virusa Baill (roots);

Icacinaceae: Pyrenacantha caurabassana Baill (tuber, green fruits); Labiatae: Hoslundia opposita Vahl (leaves), Leonotis nepetaefolia R. Br. (plant);

Lauraceae: Cassytha piliformis L. (plant);

Leguminosae: Acacia mellifera Vehl. (bark), A. robusta Burch. (root bark), A. senegal Willd. (roots), Adenanthera pavonina L. (seeds, leaves), Bauhinia reticulata DC. (plant), Caesalpinia pulcherrina Swartz. (flowers, rootbark), Cassia fistula L. (bark), C. obtusifolia L. (plant), C. occidentalis L. (plant), Desmodium sp. (plant), Dichrostachys cenerea Wight. Arn.(stem), Peltophorum petocarpum K. (roots, bark), Pongamia pinnata P. (leaves and rootbark), Pterocarpus angolensis DC. (bark), Stylosanthes fruticosa Alston. (plant);

Liliaceae: Asparagus sp. (plant);

Loganiaceae: Strychnos madagascarensis Poir. (root bark);

Malvaceae: Malvastrum coromandelianum Garcke. (plant), Sida cordifolia L. (roots), S. serratifolia L. (plant), S. spinosa L. (roots, leaves);

Ochnaceae: Brackenridgea Zanguebarica Oliv. (root bark);

Rhamnaceae: Zizyphus pubescens Oliv. (stem);

Solanaceae: Withania somnifera Dun. (plant),

Sterculiaceae: Dombeya shupangae K. Schum. (bark, leaves), Melhania velutina Forsk. (leaves), Waltheria indica L. (leaves);

Tiliaceae: Corchorus olitorius L. (fruits and seeds), Grewia stuhlmannii K. Schum. (roots), Triumfetia rhomboidea jacq. (bark and roots);

Verbenaceae: Lantana camara L. (leaves), Vitex sp. (plant, roots);

Vitaceae: Cissus rotundifolia Vahl. (leaves),

Table 5: Zones of inhibition of bacterial growth (nun) by (+)-b-senepoxide and (+)-pandoxide

Bacteria	Zones of inhibition (diameter)
	(+)-b-senepoxide	(+)-pandoxide
Escherichia coli	29	20
Staphylococcus aureus	20	16
Klebsiella pneumoniae	23	15.5
Pseudomonas aeroginosa	20	20
Bacillus subtilis	22	22
Salmonella typhi	21	19

Both the compounds showed bacteristatic activity and no bactericidal properties. (+)-b-Senepoxide showed a minimum inhibitory concentration (MIC) of 62.5 mg/ml.

Chart 1: Some antibacterial compounds from Tanzanian medicinal plants.

2

3

4

5

6

7

8

9

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11

Identification of clovanediol: A rare sesquiterpene from the stem bark of canella winterana L. (Canellaceae), using spectrophotometric methods

D.W. KIOY,* A. I. GRAY,** and P. G. WATERMAN**

* Kenya Medical Research Institute (TMDRC),
P.O. BOX 54840, Nairobi, Kenya.

**Phytochemistry Research Laboratories,
Department of Pharmacy, University of Strathclyde
Glasgow G1 IXW, U.K.

ABSTRACT

The Canellaceae is a small plant family found in continental Africa, Madagascar and America. In Kenya the two species (Warburgia ugandensis and W. stuhlmannii) that belong to this family are used traditionally as medicines against many aliments. Canella winterana are trees with an aromatic and pungent bark, found in Florida and the West Indies. Its stem bark has been used as a flavouring agent, as spices and as medicine. Previous investigations of the plant have reported the occurrence of monoterpenes, sesquiterpenes, phenylpropanoids and mannitol in the plant. In a re-investigation of the plant, ground stem bark was macerated with petrol, ethyl acetate and methanol. The separation of the extracts chromatographically, that is, column chromatography, vacuum liquid chromatography and high performance liquid chromatography (HPLC) etc., yielded a number of compounds. Of these compounds, one was identified as clovanediol, with the help of nuclear magnetic resonance (NMR), infrared (IR), ultraviolet (UV).

Introduction

The Canellaceae is a small plant family of glabrous, aromatic trees and has been described (Good, 1971 and 1974) as a discontinuous family occurring in America, Africa and Madagascar. The Warburgia species are found in East and Central Africa, and are used traditionally as medicines and spices (Kokwaro, 1976; Watt and Breyer-Brandwijk, 1962; Dale and Greenway, 1961). Canella is a genus consisting of one species, C. winterana and is found in Southern Florida, through the Caribbean, and in Colombia (Hutchinson, 1964). It has been used traditionally as a spice and as medicine (BPC, 1934).

Earlier investigations of the stem bark of Canella reported the occurrence of monoterpenes, eugenol and mannitol (Claus, 1956 and Gibbs, 1974), drimane sesquiterpenes [canellal = muzigadial], 3-methoxy-4, 5-methylenedioxycinnamolide (El-Feraly, 1978 and 1979), and 4, 13-a-epoxymuzigadial (Al-Said et al., 1989). During our re-investigation of the stem bark, we reported on the isolation and identification of myristicin, eugenol, warburganal, mukaadial and 9a-hydroxycinnamolide (Kioy et al., 1989). We now report on the further identification of a tricyclic sesquiterpene, clovanediol (Aebi et al., 1953), using spectroscopic methods.

Materials and method

Plant material

The stem bark of Canella winterana was collected from the coastal bluffs at East End Grand Cayman (Kenya) in August 1981.

Extraction and isolation

Ground stem bark (85 g) was macerated in the cold using petroleum ether (boiling range 40-60°), ethyl acetate, and methanol, in succession. Comparative thin layer chromatography (TLC) of ethyl acetate and methanol extracts showed similar chromatogams, and they were mixed together and separated by means of Vacuum Liquid Chromatography (VLC). Silica gel (Merck, 60 G) chromatography (chloroform, and then a gradient of chloroform and methanol) gave a fraction which contained one major compound. This was purified by HPLC eluting with methanol/chloroform (2:100 v/v) and then by preparative HPLC to yield 18 mg of pure clovanediol.

Physio-chemical measurements

Melting points were determined using a Reichert sub-stage microscope melting point apparatus, and are uncorrected. Specific rotations, [a]_D were measured using a Perkin-Elmer model 241 polarimeter. The infra-red (IR) spectrum was recorded as a KBr disc on a Perkin-Elmer model 781 infra-red spectrophotometer. The Proton Nuclear Magnetic Resonance (¹H-NMR) spectrum was recorded on a Bruker WH-360 operating at 360 MHz instrument, and the carbon-13 nuclear magnetic resonance (¹³C-NMR) spectrum was recorded on a Bruker WH-360 instrument operating at 90.56 MHz. High resolution electron impact mass spectral data were obtained on an AEI-MS 902 double focussing instrument by direct probe insertion.

Discussion

The structure of clovanediol was established on the basis of the spectral data, and eventual comparison with literature information. Accurate mass measurements gave the molecular ion at m/z 238, which is consistent with formula C₁₅H₂₆O₂. The (¹³C-NMR) spectrum contained 15 carbon resonances, while Distortionless Enhancement by Polarisation Transfer (DEPT) experiments revealed that these consisted of three methyl, six methylene, three methine and three quaternary carbons.

Combined spectroscopic analysis and extensive single frequency irradiations and nuclear overhauser enhancement (NOE) experiments ultimately established that the isolated compound was clovanediol.

The relative stereochemistry was established by considering the magnitudes of the coupling constants, and by NOE experiments. The melting point was in agreement with the previously reported value of 152-153° (Aebi et al., 1953). This, together with the specific rotation of +6° [reported: +5° (Aedi et al., 1953)], confirmed the structure of clovanediol.

Scheme 2.4b: Proposed fragmentation pattern for clovanediol (Gupta and Dev. 1971)

Conclusion

The most logical approach towards the discovery of new drugs is through investigation of medicinal plants. This paper discusses an example on how compounds isolated from medicinal plants are identified. Although different physical-chemical methods may be used, the steps outlined in this paper are essential. In some plants, the active compounds are present in very small amounts which would otherwise be difficult to be investigated using other methods. But the use of modern spectroscopic methods has made it possible to carry out complete identification of compounds, even when they are in minute amounts.

The biological activity of clovanediol has not been investigated. However, it would be interesting to see if this compound has any activity.

References

Aebi, A., Barton, D. H. R. and Lindsey, A. S. (1953): J. Chem, Soc. (C): 3124.

Al-Said, M. S. Khalifa, S. I. and El-Peraly, F. S. (1989): Phytochemistry 28: 297.

BPC (1934): 238.

Claus, E. P. (1956): Pharmacognosy, Henry Kimpton, London.

Dale, I. R. and Greenway, P. J. (1961): In Kenya trees and Shrubs, Hatchards. London: 654.

El-Feraly, F., McDhal, A. T. and Onan, K. D. (1978): J. Chem. Soc. Chem. commun.: 75.

El-Feraly, F. S. and Hofftetter, M. D. (1979): J. Nat. Prod. 43: 407.

El-Sherei, M. M., El-Feraly, F. S., El-Sohly, M. A and Stanford, D. F. (1987): Fitoterapia 58: 272.

Gibbs, R. D. (1974): Chemotaxonomy of flowering plants, 2. McGill-Queens University Press, Montreal: 783.

Good, R. (1971): The Geography of flowering plants, 3rd Edition. William Cloves & Sons Ltd. London: 63.

Good, R The Geography of flowering plants, (1973) 4th Edition. William Cloves & Sons Ltd. London: 64.

Gupta, A. S. and Dev, S. (1971): Tetrahedron 27: 635.

Hutchinson, J. (1964): The genera of flowering plants 1. Oxford University Press: 62-65.

Kioy, D., Gray, A. I. and Waterman, P. G. (1989): J. Nat. Prod. 52: 174.

Kokwaro, J. O. (1976): Medicinal Plants of East Africa. East Afr. Lit. Bureau, Nairobi: 45

Watt, J. M. and Breyer - Brandwijk, M. G. (1962): Medicinal and poisonous plants of Southern and Eastern Africa, E. S. Livingstone Ltd., Edinburgh & London.

Williams, D. H. and Fleming, I. (1980): Spectroscopic Methods in Organic Chemistry, 3rd Edition, McGraw Hill Co. (UK) Ltd.

A comparative study of the traditional remedy ''Suma-kala'' and chloroquine as treatment for malaria in the rural areas

NOUHOUM KOITA

The Clinical Section
Traditional Medicine Division
P.O.Box 1746, Bamako, Mali.

Introduction

Traditional medicine has been utilised by the majority of the World population for thousands of years. Until the beginning of the 19th century, all medicine was traditional (Jellife, 1977). Yet in many developing countries it is true that for the majority of the rural population traditional medicine is the only primary or any other kind of health care available (Heggenhougen et al., 1988). For more than 70% of the population in Africa, traditional medicine is the first, if not the only health care system available in the poor rural and urban areas. In recognition of this fact, the World Health Organization underlined the potential role that traditional medicine may play in reinforcing the health care system through the primary health care approach in developing countries (W.H.O., 1978). The value of traditional medicine may be relative to both its pharmacological and/or biomedical value, as well as its psychological and social values (Heggenhougen et al., 1988).

Medicinal plants and their products have been used in the treatment of malaria throughout the tropics and subtropics. Such experience is not to be ignored. Instead, it should be actively investigated so that basic information can be made available for the preparation of standardized, effective and non-toxic remedies. Quinine, from the bark of Cinchona, whose legend dates from the 17th century (Bruce-Chawatt, 1985; Phillipson, et al., 1986) is an outstanding example of a plant product which has been used for centuries in the treatment of malaria. The Chinese antimalarial, quinghaosu, is another example of this kind. The Ministry of Health in Mali has been trying to study the resources of Malian traditional medicine, with emphasis on the evaluation of the effectiveness of its medicinal plants.

The main purpose of this paper is to analyse and discuss the results of a clinical trial which compares a Malian traditional remedy called "Suma-Kala", with chloroquine, as a treatment for malaria in the rural areas of Mali.

Materials and Methods

A randomized controlled trial of "Suma-Kala" in the treatment of malaria was carried out at the Selingue Health Center, from July to September, 1987.

Preceding the main study, a two weeks training and pilot study took place in the Selingue Health Centre, attended by all personnel involved in the study. The aims of these were to review and standardize the clinical and laboratory techniques and also to test and correct the material and methodology. The ethical problems of the study were discussed. Any complicated case was to be admitted immediately to the health centre for proper management.

Preparatory visits were paid to the local authorities by the doctor of Selingue Health Centre and his team, to explain the objectives of the study, and to ask for their approval and cooperation.

Objectives of the study

The objectives of the study were:

(a) to confirm the antimalarial property of the "Suma- Kala";
(b) to assess the acceptability and tolerance of the "Suma- Kala"; and
(c) to compare its activity with that of a well established standard antimalarial, which, in our case, was chloroquine.

Study area

The study was conducted in four villages in the Selingue area during the rainy season, from July to September in 1987 (Figure 1). Selingue is the National Institute of Public Health Research's rural health centre, in a dam area which deals with water-related diseases. Selingue is 135 km from Bamako. The background information about Selingue area is adapted from Traore (1986).

Human population and randomization

The method used consisted of a randomized control, and partially blind clinical trial. The four villages closest (from 3 to 10 km) to the health centre were chosen for a good follow up and case management in the event of complications. The chief and the health committee of each village chose the place (rooms) where the examination of the patients took place. All patients who thought had "sumaya" (malaria fever) were invited to attend the clinical examination.

All the patients were randomized on their arrival on day 0 and treated. The patients were randomly allocated to the "treatment" group ("Suma-Kala") or the "control" group (chloroquine) alternatively in a group of 10 on arrival. The method of "tossing a coin" was used to decide the order of allocation For the purpose of this study, the patients were told that they were receiving traditional remedies made by the Traditional Medicine Division of their own country by their own countrymen. The treatment was administered on an outpatient, basis. Neither the patient nor the medical team was blind to the treatment since the chloroquine was administered in capsules while the "Suma-Kala" was given as a decoction.

The patients were also questioned on the first day (day 0) about the recent prophylaxis and treatment and their urine was tested for presence of detectable concentration of chloroquine, amodiaquine, quinine, quinidine or mefloquine using the Dill-Glasko test.

A positive result of the Dill-Glasko test excluded the patient from the study. Any patient younger than 5 years and any pregnant woman was excluded.

The following conditions also excluded patients from the study:

(a) parasitaemia less than 5000 malaria parasites per cubic mm of finger blood smears;

(b) serious illness conditions such as, liver and kidney failures, acute or chronic pneumonia, hepatitis, and allergy;

(c) presence of serious digestive troubles such as, diarrhoea, intensive vomiting; and

(d) signs of dehydration.

Fig. 1: Map of Mali

Two teams were responsible for the study: one team in the field was in charge of the clinical examination and the blood film preparation without knowing the parasitaemia progress; and the second examined parasitaemia in the laboratory in Selingue Health Centre, without knowing which drug the patient had received. Only the result of the first blood smear (day 0) was communicated the following day (on day 1) to the clinical examination team for exclusion from the study of any patient with a parasitaemia less than 5000 of parasites per cubic mm on the first day (day 0). The other results of parasite count (days 1,3,5,7,14 and 21) were kept secret by the head of the laboratory team until the end of the study. On the other hand, the laboratory team members could not distinguish whether a slide they were examining belonged to a patient under the new drug or not.

Preparation of the drug

Both treatments were administered orally and were continued for seven consecutive days. The chloroquine diphosphate was made by our partners in France for the purpose of the study and presented in 100 milligramme and 300 milligramme capsules. Empty placebo capsules similar to those of chloroquine were made in France and sent to us by "CREDES, Terre des Hommes".

"Suma-Kala" was analysed botanically, chemically and pharmacologically. The detail on its botanical, chemical and pharmacological preliminary studies are available elsewhere in the Traditional Medicine Division in Bamako, Mali (Study of "Suma-Kala" presented to the 1988 meeting of the Scientific Committee of the National Institute of Public Health Research in Bamako).

The "Suma-Kala" is composed of three medicinal plants including Cassia occidentalis L. (locally known as: Mbala mbala), Lippia chevalieri Moldenke (locally known as: Kaniba djan); and Silanthus oleraceae Jacq. (locally known as : Mame - Farimani) (Figures 2, 3 and 4).

"Suma-Kala" was prepared by the galenic section of the Traditional Medicine Division in its laboratory in Bamako, Mali. It was a mixed powder of the leaves of Cassia occidentalis L. and Lippia chevalieri M. and of the flowers of Spilanthus oleracea J. It was presented in a small plastic bag each containing 10 grams of this mixture of powder with the following proportions:

Cassia occidentalis L. 64%
Lippia chevalieri M. 32%
Spilanthus oleraceae J. 4%

Although the population in rural areas in Mali are used to decoction preparation, the patients randomised to "Suma- Kala" were shown on the first day (day 0) how to prepare the decoction. Subsequently they were required to prepare the decoction for themselves daily at home. The decoction was prepared by boiling a bag of 10 grams of "Suma-Kala" in a half litre (500 ml) of water for about 15 minutes. Sugar can be added to sweeten its taste.

Dosage

The treatment was administered to the patients at home (outpatient). The chloroquine treatment was standardized and consisted of swallowing 10 milligrammes per kilogramme body weight during three consecutive days. For the remaining four days of the week, the empty placebo capsules were given so that the duration of the treatment for both drugs lasted for seven consecutive days.

The treatment with "Suma-Kala" consisted of drinking the decoction made from "Suma-Kala", twice a day, for four days, and then once a day for three days. The quantity of "Suma kala" bags and chloroquine capsules for the daily treatment were given to the patients each day after the clinical examination and the making of the blood film for parasitaemia count.

Clinical parameters

A form was used to record each patient's identity and the clinical parameters each day of examination. The biological parameter (parasitaemia) was recorded separately in the laboratory record. In addition to the identity (name, sex, age, village, date of examination, and the observer's name), the clinical record included the follow up of auxiliary temperature, headache, vomiting, shivering, nausea, and the side effects such as allergy and digestive troubles (see copy of the form in annex). These clinical parameters were recorded every day from day 0 (first day of examination) to day 7 (eighth day of examination) for assessing the curative effect of the drugs; and also on days 14 and 21 for assessing the eventual residual protective effect of the drugs.

Figure 2: Habit drawing of Cassia occidentalis

Figure 3 : Habit drawing of Spilanthus oleracea

Flgure 4: Habit drawing of lippia chevalieri

Laboratory methods

The laboratory method used was the malarial parasite count from finger thick and thin blood smears, using the W.H.O. standard techniques (W.H.O., 1984).

The labelling of the slides was carried out with a diamond pencil. The finger of the patient was cleaned with 70% ethanol. Staining of blood was done using Giemsa stain. The films for malaria parasites were collected in the field during the clinical examination on days 0, 1, 3, 5, 7, 14 and 21. All blood films collected were read in the first instance in the laboratory of Selingue Health Centre and cross checked after one month in the parasitology Department of the Medical School in Bamako. The laboratory observer teams were composed in Selingue by the two laboratory technicians under the supervision of the parasitologist head of the laboratory, and in Bamako by one laboratory technician and the physician parasitologist, head of the Parasitology Department of the Medical School.

The thick films were examined using the "farmer ploughing his field" technique: across the film to the opposite edge, and a slight lateral move, then back across the film, a slight lateral move. The process was repeated. For the thin films a "battlement" technique was used traversing the edge of the tail in short vertical and horizontal tracks. The number of parasites per 200 White Blood Cells were counted and parasite density was calculated taking 8,000 WBC per cubic mm as an average WBC count. A simple mathematical formula was used to convert the counts into the number of parasites per cubic mm of blood. For the minimum threshold, W.H.O. suggests 1000 parasites per cubic mm (W.H.O., 1984), but we decided to use 5000 parasites which is according to findings in Africa (Trape, 1985), a useful discriminant for separating children in whom malaria was thought to be the cause of their illness, from those in whom it probably was not. This was because of the fact that most patients in endemic areas of malaria like Selingue, could have a parasitaemia up to 1000 per cubic mm without showing the clinical signs of malaria. Therefore, any patient with a parasite density less than 5000 per cubic mm was excluded from the study.

Data analysis and reports

It was planned to carry out a computer analysis of the data and also at the Statistics Unit, the National Institute of Public Health Research in Bamako, using appropriate statistical tests (Z-test or t-test or Mantel-Haenszel test) to compare the effects of the two drugs. The results were supposed to be diffused at the different levels of utilisation.

Results of the clinical trial

About 3000 people presented with "sumaya" were included in the study. All were randomised on their arrival, then examined and their complaints were examined and treated if necessary. However, according to the criteria for inclusion in the study, only 53 of these patients were eligible for comparing "Suma-Kala" and chloroquine from July to September 1987 in Selingue. Thirty-six of the patients belonged to the "Suma-Kala" treated group and 17 belonged to the chloroquine treated group.

Age and sex distribution

The age and sex distribution is shown in Table 1. The study population was very young: 70% of the 53 patients were under 10 years. Only one patient was older than 25 years (she was 45 years old). Fourty-five percent of the patients were males and 55% were females. The results of the Mantel-Haenszel test have shown that the sex difference between the two groups after allowing for age group was not statistically significant (c² = 0.030, with a degree of freedom = 1 and p > 0.05). On the other hand, the age group difference between the "Suma-Kala" treated group and the chloroquine treated group controlling for the sex was not significant (Mantel Haenszel c² = 0.030 with df = 1 and p > 0.05).

Table 1: Age and sex distribution of the study population per treatment

Age group	Suma-Kala			Chloroquine
(years)	Males	Females	Total	Males	Females	Total
5-9	13	11	24	5	8	13
10-14	2	4	6	1	2	3
15-49	2	4	6	1	0	1
TOTAL	17	19	36	7	10	17

Follow-up of the study population

Table 2 shows the follow up of the study population per day and age group. The overall proportions of drop-out before the end of the study were similar among the two groups and concerned mainly the first age group (5-9 years). Eighty-six percent of the patients in the "Suma-Kala" group completed the treatment, while 71% in the chloroquine group completed the treatment. Therefore, the follow up was 15% better in the "Summa-Kala" group on day 7. However, this difference between the proportion of patients who completed both treatments was not statistically significant (Z = 0.064, p = 0.95).

Table 2: Follow up of the treatment by the patients per day and per age group

"Suma-Kala" treated group							Chloroquine group
Days	5-9	10-14	15-19	24-25	45-49	Total	5-9	10-14	15-19	Total
0	24	6	3	2	1	36	13	3	1	17
1	24	6	3	2	1	36	13	3	1	17
3	23	6	3	2	1	35	10	3	1	14
5	20	6	3	2	1	32	8	3	1	12
7	19	6	3	2	1	31*	8	3	1	12*
14	18	5	3	2	1	29	8	3	1	12
21	17	4	3	2	1	27	6	3	1	10

Clinical parameters

Figures 5 and 6 show the proportions of patients who became free of the clinical parameters per treatment and per day of treatment. The comparison of the effects of the two drugs on the clinical parameters show that "Suma-Kala" was as effective as chloroquine, if not better.

We considered an auxiliary temperature of 37.5°C or higher as fever according to findings in Africa (Greenwood et al, 1987; Delfini, 1968; Cobban, 1960). The proportions of patients who had fever, headache, shivering, nausea and vomiting at the start (on day 0) and became free of these clinical parameters after 7 days of "Suma-Kala'' treatment were respectively 59.3 %, 76%, 62.5 %, 93.3%, and 79%, while the proportions of patients free of clinical parameters under chloroquine treatment were respectively 50%, 47%, 80%, 75%% and 68%. The general trend was suggesting a better improvement under "Suma-Kala". However, the difference of the effects of the two drugs against fever, headache, shivering, nausea and vomiting was not statistically significant (all p > 0.05). The same trend of better improvement of the clinical parameters on days 14 and 21 was noticed, but the difference was not statistically significant (p > 0.05).

Side effects

No clinical complication was noticed during the follow up of the patients in spite of the high parasitaemia at the start of the treatment. Few side effects were noticed. Three cases of allergy to chloroquine causing the treatment to be abandoned on day 3 were noticed among the chloroquine group, while none was reported among the "Suma-Kala" group. One case of constipation was declared among the "Suma-Kala" group, and none among the chloroquine group.

The "Suma-Kala" was very well tolerated by the patients. Table 3 shows the proportion of patients developing clinical parameters later, on days 3, 5 or 7, without having them at start. The differences between these proportions using Fisher's exact test were not statistically significant for all (p > 0.05), except for the allergy in which case "Suma-Kala" was better than chloroquine (p < 0.05).

Table 3: Proportion of patients developing clinical parameters later on days 3, 5 or 7 without having them at start

Parameters	"Suma-Kala" group	Chloroquine group
Fever	2/9 (22.2%)	1/7 (14.3%)
Headache	1/3 (33.3%)	1/2 (50%)
Shivering	1/4 (25%)	2/5 (40%)
Nausea	0/5 (0%)	0/13 (0%)
Vomiting	0/22 (0%)	0/14 (0%)
Allergy	0/36 (0%)*	3/17 (18%)*
Indigestion	1/36 (3%)	0/17 (0%)

* p < 0.05 using Fisher's exact test.

Biological parameters

Plasmodium falciparum was the only type responsible for the malaria infection in our study. The results of the biological parameters are shown in Tables 4 and 5 and in figure 7. These results suggested that chloroquine was more effective than "Suma-Kala" in cleaning the parasites of malaria from the finger blood smears.

Figure 5: Proportion of patients who had fever on day 0 and became free from it later per day and treatment

Figure 6: Proportion of patients with headache on day 0 but none later on per day and per treatment

Figure 7: MEAN LOG (PARASITAEMIA .1) PER DAY AND PER TREATMENT

Before the treatment started (on day 0), the overall geometric mean of parasite count was 17975.3 among the "Suma-Kala" group for a population of 36 patients, while among the chloroquine group it was 13414.3 for a population of 17 patients. The difference between the parasitaemia of the two groups using mean log (parasitaemia count + 1) was not statistically significant (t-test = 1.37, df = 51, and p > 0.05).

At the end of the 7 days treatment, the geometric mean of parasitaemia count became 153.8 among the "Suma-Kala" group for 31 patients, while in the chloroquine group it became 3.4 for 12 patients. The difference between the means of parasitaemia using the mean log (parasitaemia count +1) became statistically significant (t = 2.98, df = 41 and p < 0.05) (Table 4). These figures in Table 4 were suggesting that chloroquine was better than "Suma-Kala" in cleaning the malaria parasitaemia at the end of both treatments.

Table 4: Mean log (parasitaemia count + 1) and geometric means per day and per treatment

	"Suma-Kala" group			Chloroquine group
Days	log(cnt+1)N	SD	Geom. mean	log(cnt+1) N	SD	Geom. mean
0	4.2547 36	.3079	1795.3	4.1276 17	.3274	13414.3
1	3.8774 36	.6282	7539.5	3.0301 17	.9380	1046.4
3	2.9449 36	1.4131	880.0	1.3995 13	.9204	24.1
5	2.4817 32	1.4201	302.2	.7175 12	.8457	4.2
7	2.1898* 31	1.6239	153.8	.6417* 12	1.2096	3.4
14	2.3245 28	1.6530	219.1	.1165 12	.4036	3.0
21	1.8449 26	1.6094	69.0	.5454 10	.8921	2.5

* p > 0.05

Residual effect of the drugs

We expected the protective ("prophylactic" or residual) effect of "Suma-Kala" to be continued one or two weeks after the treatment like the way it usually happens with chloroquine. The data on days 14 and 21 (Tables 4 and 5) were for the study of these eventual residual effects.

The residual effects of "Suma-Kala" against the clinical symptoms of malaria were similar with those of chloroquine. However, the parasitaemia of 5 among the 18 patients of the "Suma-Kala" group with low parasitaemia (less than 1000 parasites per cubic mm) on day 7 (end of both treatments), became high (greater than 1000 parasites per cubic mm) on days 14 and 21, while the patients of the chloroquine group with low parasitaemia on day 7 remained with a low parasitaemia. The difference between these proportions (13/18 and 12/12 or 10/10) was statistically significant (using Fisher's exact test p < 0.05). Although the sample size was small, this result was suggesting that the residual protective effect of "Suma-Kala" was less than that of chloroquine on days 14 and 21.

Table 5 shows the proportions of patients with parasitaemia less than 100 per cubic mm per day and per treatment. At the end of the treatment (day 7), the difference between these proportions (58.1 % versus 92%) was not statistically significant (Z = 1.72 and p = 0.085 > 0.05).

On day 21 also, the proportions of patients with a parasitaemia less than 100 per cubic mm (63 % versus 100%) were not statistically significant (Z = 1.81, p = 0.07 > 0.05).

Table 5: Proportions of patients with parasitaemia less than 1000 per cubic mm per day and per treatment

Days	"Suma-Kala" group	Chloroquine group
0	0/36 (0%)	0/17 (0%)
1	3/36 (8.3%	10/17 (59%)
3	15/35 (43 %	13/13 (100 %)
5	17/32 (53.1 %	12/12 (100 %)
7	18/31 (58.1 %)	11/12 (92%)*
14	16/29 (55.2 %)	12/12 (100 %)
21	17/27 (63 %)*	10/10 (100 %)*

* p > 0.05

Discussion of study design and results

A good study design should be made in such a way that any observed difference between the treatment and the control group can be attributed to the real effect of the treatment.

We thought of using the randomized double blind placebo control trial but unfortunately, the drug section could not make placebo for the "Suma-Kala", and it was impossible to make the leaves composing "Suma-Kala" unrecognizable. On the other hand, because of ethical consideration, a control group receiving no treatment or placebo was not thinkable. We finally ended up with a randomized control blind method.

Whenever possible, it is preferable that neither the participant nor the investigator knows which treatment has been received until after the end of the trial.

For future trial if the drug section is able to make a placebo preparation indistinguishable to "Suma-Kala", then we can achieve a double blind method by giving to the control group chloroquine capsules plus a placebo decoction and to the treatment group "Suma-Kala" decoction plus placebo capsules. We randomized alternatively by group of 10, the patients declaring having "Suma-Kala" (malaria) on their arrival to the control and the treatment group. The method of "tossing a coin" was used to decide the order of allocation. The patients were unknown by the examiners and therefore this limited the selection bias. However, the randomization method we used could be improved for instance by randomizing only the eligible patients and by using random number tables with odd numbers (1,3,5,7,9, etc.) corresponding to the chloroquine treated group and even numbers (0,2,4,6,8, etc.) to the "Suma-Kala" treated group (or vice versa).

Instead of numbers, different combinations of letters could also be used to randomize the patients (i.e., AABB, ABBA, ABAB, BBAA, etc., where A is for the control group and B for the treatment group, or vice versa). The order of allocation should be preferably decided before the start of the trial. It is sometimes also desirable to arrange the allocation so that equal numbers of participants will be entered into each group. That is what (Kirkwood (1988) called "restricted randomization" or "randomization with balance."

We had a lot of trust on the patients for following correctly the instruction for the preparation of "Suma-Kala."

The urine of the patient became positive to the Dill- Glasko test, like it happened with chloroquine. Therefore Dill-Glasko test was not performed after day 0 to check whether patients on "Suma-Kala" also gave themselves chloroquine. This could perhaps be avoided by doing the study on patients basis in which case one would have to think about selection biases. For future trials we should find a way to perform spot check.

There is no perfect approach and the field conditions are such that compromise between theory and reality is obligatory. What is essential is to be as close as possible to the ideal as the conditions permit it. The fact that our population was very young, may be due to the high level of our threshold of 5000 parasites per cubic mm, which is easier to get among the young people because the older people in a high endemic area may have already developed their semi-immunity to malarial infection (Bruce-chawatt, 1985; Trape, 1985).

Because of the conditions required for the inclusion in the study, our sample collected during three months seems small. Most of the people of Selingue very often take chloroquine in all cases of fever or for prophylactic purpose. Therefore, the urine in most of the people was positive for the Dill-Glasko test. In these conditions, a reasonable sample size was difficult to obtain in three months and the resources available could not permit a longer stay.

The results so far of the study are interesting for several reasons. To our knowledge this study is perhaps one of the first (if not the first) clinical trials comparing African traditional antimalarial medicinal plants and allopathic antimalarial drugs.

Some preliminary work has been done in some countries but not published (Bray, personal communication). Most of the studies published referred to in vivo experiments on mice infected berghei (Makinde and Obih, 1984; Peter, 1970) or in vitro (Phillipson et al., 1986).

The plants which composed "Suma-Kala" were known in West Africa and used in traditional medicine against several diseases.

Cassia occidentalis L. was the most popular, and the most used by the traditional practitioners mainly against malaria fevers, headache and skin diseases (Kheraro, 1974; Ayensu, 1978; Rozat, 1979; Oliver, 1986, Sofowara, 1982).

The findings have shown that C. occidentalis has antiparasitic and antibacterial activities (Oliver, 1986). Spilanthus oleraceae J. was known too, and used as a medicinal plant in West Africa (Kheraro, 1974; Rozat, 1979). The extracts of its flower-heads killed Anopheles larvae and the whole plant has shown insecticidal properties (Oliver, 1986).

Lippia chevalieri M. was the least popular among the plants which composed "Suma-Kala." However, it was also used as a medicinal plant in West Africa (Kheraro, 1974; Rozat, 1979).

The results of the study have shown that "Suma-Kala" is as effective as chloroquine against the symptomatic signs of malaria. Therefore, the traditional practitioners are quite right in using these medicinal plants against malaria because their diagnosis and prognosis of the disease are mainly based on clinical symptoms.

The current dosage of "Suma-Kala" was less fast than that of chloroquine in suppressing the malaria parasitaemia. The difference between the proportion of patients with parastitaemia less than 1000 parasites per cubic mm, was not statistically significant at the end of the treatment (day 7). However, the difference between the mean log (parasitaemia count + 1) of the two groups was statistically significant. Therefore, chloroquine was more effective than "Suma-Kala" in clearing the parasitaemia.

A parasitaemia of 1000 per cubic mm of finger blood smears is common among the people living in endemic areas like Selingue, and is well tolerated. That is why WHO suggested it as a cut off point (WHO, 1984) and (Trape 1985) suggested a higher level of minimum threshold of 5000. If we consider a parasitaemia of 1000 per cubic mm as "normal" in endemic areas during a period of high infection (raining season) as suggested by many authors (W.H.O., 1984; Trape, 1985; Greenwood et al., 1987), "Suma-Kala" and chloroquine could be considered as having similar effects against malaria, because the difference between the proportion of patients among the two groups having a parasitaemia less than 1000 parasites per cubic mm was not significant at the end of treatment.

It could be interesting to compare the effect of "Suma- Kala" to that of a placebo. But for ethical reasons, a placebo group was not used in our study, and in the literature we did not see any publication referring to placebo effect on malaria. Nevertheless, no case of complication was noticed among our patients in spite of very high parasitaemia cases (some were as high as 80,000 parasites per cubic mm). Furthermore, the difference between the biological parameters (proportion of patients with a parasitaemia less than 1000) on one hand, and between the clinical parameters (proportion of patients with fever, headache, vomiting, nausea, shivering) on the other hand, were not statistically significant. And also, the likely pattern of high parasitaemia in untreated malaria would be the occurrence of clinical malaria with a certain number of complications (for instance convulsions in children) and probably some cases of deafness. Therefore, we were convinced that the effect of "Suma-Kala" was far more than that of a placebo.

The two drugs were well tolerated, but "Suma-Kala" was better tolerated than chloroquine. This was illustrated by three facts. Firstly, the study did not show any side effect from "Suma-Kala", while 3 patients among the chloroquine treated group abandoned the treatment on the third day because of the allergy to chloroquine that they developed. Secondly, the difference between the proportions of patients who have developed later on clinical parameters without having them at start was not statistically significant, except for the allergy to chloroquine noticed among the chloroquine group. Thirdly, the follow-up of the treatment by the patients was 15 % higher among the "Suma-Kala" group.

An attempt was made to measure the protective (residual or "prophylactic") effect of the drug by looking at the clinical and biological (parasitaemia) parameters on days 14 and 21. The interpretation of the data on days 14 and 21 was difficult, because of the size of the sample becoming smaller on one hand, and on the other hand, these data are rather related to the eventual residual protective effect than to the prophylactic effect. Nevertheless, the two drugs seemed to have the same residual protective effect against the clinical parameters, but concerning the biological parameter, "Suma-Kala" seemed to have a less protective effect against malaria reinfection than chloroquine.

Conclusion

Research into medicinal plants should not stop because the herbal medicine still has an immense potentiality to enrich the universal pharmacopoeia.

Although the cooperation between allopathic and traditional medicine is not easy to build, it is necessary because it represents a valuable national resource for many developing countries. Therefore, it should be taken into account for the achievement of the World Health Organization goal of an ideal health for all. In Mali, as elsewhere, the research into traditional medicine should be extended beyond the focus on phytotherapy, and efforts should be made to do more research in the other aspects of traditional medicine because they heal people, even though they may not cure disease. The Malian traditional antimalarial remedy, "Suma-Kala", is working. The study showed that it is as efficient as chloroquine against the clinical symptoms such as fever, headache shivering, vomiting, and nausea, and also that it was better tolerated. However, "Suma-Kala" was not as fast as chloroquine in clearing malaria parasitaemia.

More research should be done in order to improve the mode of administration of "Suma-Kala", and to increase its speed of clearance of malaria parasitaemia. For instance, more research on its dosage and galenic presentation could improve its effects. Although the study has shown interesting results, its design, particularly the sampling method and the 'blindness', could be improved for future clinical trials. A randomised blind control trial could be applied, provided that a placebo for the "Suma-Kala" is available, so that the "control group" will receive chloroquine capsules (or the standard treatment) plus placebo (decoction), and the "treatment group" will receive "Suma-Kala" (or the new drug), plus placebo (capsules).

Meanwhile, the production and commercial exploitation of "Suma-Kala" in Mali should not be delayed. The plants which compose "Suma-Kala" are locally available and could also be locally cultivated. Therefore, the production of "Suma-Kala" should be regarded in the perspective of reducing the burden of drug importation, and also as a potential alternative source of income for the peasants.

Acknowledgements

I am grateful to Prof. Mamadou Koumare for the initiation of contemporary research into traditional medicine in Mali; Ms Gillian Maude for providing invaluable assistance and encouragement in preparing this paper; Drs. Kris Heggenhougen, Thierry Mertens and Dorothy Bray for reading early drafts and giving useful feedback; Drs. Drissa Diallo, Ogobara Doumbo, Moctar Guindo, the people and the personnel of the Selingue Health Centre, and the personnel of the Traditional Medicine Division for actively taking part in designing and implementing the study; Dr. Martin Vitte and her colleagues from "CREDES, Terre des Hommes, France" for funding of the study and the British Council for sponsoring my MSc. course in England.

References

Ayensu, E.S. (1978). Medical Plants of West Africa, Reference Publication, Inc., Michigan, U.S.A: 72-75.

Bray, D.H. Personal Communication, Department of Medical Parasitology of the London School of Hygiene and Tropical Medicine. Keppel Street, London WC1E 7HT, UK.

Bruce-Chwatt, L.J. (1985). Essential Malariology, William Heinemann Medical Books, Second Edition, London.

Cobban, K. McL (1960). Journal of Tropical Medicine and Hygiene, 63: 233-237.

Delfini, L.F. (1968). The Relationship Between Body Temperature and Malaria Parasitaemia in Rural Forest areas of Western Nigeria. W.H.O. Report WHO/MAL 68.654 [Unpublished document].

Greenwood, B.M. et al (1987). Mortality and Morbidity From Malaria Among Children in a Rural Area of the Gambia, West Africa. Transaction of the Royal Society of Tropical Medicine and Hygiene. 81: 478- 486.

Heggenhougen, K. et al (1988). Traditional Medicine and Primary Health Care. EPC Publication No. 188. London Shool and Tropical Medicine, Keppel Street, London WC1E 7HT UK.

Imperato, P.J. (1981). Modern and Traditional Medicine: The case of Mali. Annals of Internal Medicine, 95. No. 5.

Jelliffe, D.B. and Jelliffe, E.F.P. (1977). The Cultural Cul-de-sac of Western Medicine. Transactions of the Royal Society of tropical Medicine and Hygiene 71 (4): 331-334.

Kheraro, J. (1974) . La Pharmacopee Senegalaise Traditionnelle. Plantes Medicinales and Toxiques, ed. Vogot Freres, Paris.

Kirkwood, B.R. (1988). Essentials of Medical Statistics. Sackwell Scientific Publications, Oxford.

Makinde, J.M. and Obih, P.O. (1984). Screening of Morindo lucida Leaf Extract for Antimalarial Action on Plasmodium berghei berghei in mice. African Journal of Medicine and Social Science. 14: 59-63.

Oliver, B. (1986). Medicinal Plants in Tropical West Africa. Cambridge University Press, UK.

Peter, W . (1970). Chemotherapy and Drug Resistance in Malaria. Academic Press, London.

Phillipson, J.D. and O'Nell, M.J. (1986). Antimalarial drugs from plants? Parasitology Today. 2 No. 12: 355-359.

Rozat, T.A. (1979). Plantes Medicinales du Mali. Bamako, Mali.

Sofowora A. (1982). Medicinal Plants and Traditional Medicine in Africa. J. Wiley and sons Limited, Chichester.

Traore M.S. (1986). Schistosomiasis in Selingue. A Man-Made Lake in Mali. A dissertation for the MSC, C.H.D.C. London, School of Hygiene and Tropical Medicine, UK.

Trape J.F. et al. (1985). Criteria for diagnosing clinical malaria among a semi-immune population exposed to intense and perennial transmission. Transactions of the Royal Society of Tropical Medicine and Hygiene, 79: 435-442.

World Health Organization (1978). The Promotion and Development of Traditional Medicine. Technical Report Series, No 622. Geneva.

World Health Organization (1984). Advances in malaria chemotherapy. Technical Report Series No. 711.

Ethnobotany and conservation of medicinal plants

R.L.A. MAHUNNAH and E.N. MSHIU

Traditional Medicine Research Unit
Muhimbili Medical Centre
P.O. Box 65001
Dar es Salaam, Tanzania

ABSTRACT

Plants are indispensable to man for his livelihood. This paper presents the value of ethnobotany to the search for new biomedical compounds in the tropics. The general values of the rich tropical vascular plant flora as sources of direct therapeutic agents, as sources of starting points for the elaboration of more complex semisynthetic compounds, as sources of substances that can be used as models for new synthetic compounds, and as taxonomic markers for the discovery of new compounds, are highlighted. A case is made for continued research in ethnobotany, since it is estimated that 80% of the people in the Third World rely on traditional medicine for primary health care needs, most of which is plant-derived.

The whole question is addressed from socio-economic perspectives. Of all the plant-derived compounds that are used in the prescription drugs, about 50% originate from the tropics; yet it is here where the greatest threats to plant biodiversity occur. Therefore, concerted ethnobotanical research is directly linked to the urgent need for sustainable conservation programmes. It is proposed that conservation programmes for developing countries take into account both conservation of maximum plant biodiversity and focused approach aimed at individual medicinal plants.

The results should facilitate better management of our medicinal plant genetic resources for sustainable economic harvesting in both in-situ and ex-situ conservation -areas.

Introduction

Our purpose here is to urge that ethnobotanical prospecting, the exploratory process by which new useful plants are discovered, be substantially intensified. However, plant species are being lost at an ever-increasing rate, faster by orders of magnitude than rates of evolutionary replacement. Therefore intensification of ethnobotanical exploration should, of necessity, be linked to the urgent need for sustainable conservation strategies for medicinal plants since human expansionist demands can be expected to wreak environmental deterioration and biotic destruction well into the next century.

This paper specifically urges for an Increased involvement of developing nations in the exploratory and conservatory process, an involvement which, in our view, is warranted on scientific, economic and cultural grounds.

Traditional Therapy

Traditional medicine is a priceless heritage which was created in the historical course of prevention and treatment of diseases over a long period. Today, traditional systems of medicine, which utilize mostly plant-derived prescriptions, remain the source of primary health care for more than 3/4 of the Third World population. It is estimated that a third of all world pharmaceuticals are of plant origin, or if algae, fungi and bacteria are included, then two thirds of all pharmaceuticals are plant based. Therefore, traditional medicine and medicinal plants are indispensable in practice. The rich traditional ethnopharmacopoeia of the Third World's tropical flora is, indeed, indicative of the high utility of indigenous medicinal plants.

Proper development and utilization of traditional medicinal plants, is of great significance in the development of health services and provides for proper take- over of national cultures for developing countries. The merit of traditional therapeutics cannot be over-emphasized. It is easily acceptable to the community, manageable and is of low cost. With the rich traditional medicinal plant resources, efficacy of prevention and treatment of disease can be ensured by appropriate, but comparatively non-sophisticated technology and with minimal side effects. Therefore proper utilization of the traditional medicinal systems by developing nations can make significant contributions towards the implementation of the programme of Health For All by the year 2000.

Drugs from nature

Through most of man's history, botany and medicine were, for all practical purposes, synonymous fields of knowledge. Therefore, the traditional healer, usually an accomplished traditional botanist, represents, probably, the oldest professional man in the evolution of human culture. However, the advent of modern technology and synthetic chemistry has been able to reduce our almost total dependency on the plant kingdom as a source of medicine. Nonetheless, plants have traditionally served as man's most important weapon against pathogens. In fact, it seems that even the Neanderthal man knew and made use of medicinal plants.

What, then, is the value of ethnobotany to the search for new biomedical compounds? Of the hundreds of thousands of species of living plants, only a fraction have been investigated in the laboratory. This poor understanding of plants is particularly acute in the tropics. Consequently, this calls for the urgent need for continued ethnobotanical research. The importance of ethnobotanical enquiry as a cost-effective means of locating new and useful tropical plant compounds, cannot be over-emphasized. Most of the secondary plant compounds employed in modern medicine were first 'discovered' through such investigation. Some 119 pure chemical substances extracted from higher plants, are used in medicines throughout the world, and 74% of these compounds have the same or related use as the plants from which they were derived. The rosy periwinkle, Catharanthus roseus (synonymous to Vinca rosea), represents a classic example of the importance of plants used traditionally by man. This herbaceous plant, native to South-eastern Madagascar, is a source of over 75 alkaloids, two of which, vincristine and vinblastine, are used to treat childhood leukemia and Hodgkin's disease, with a significant success rate. The use of quinine from Cinchona bark to cure clinical malaria, today owes its use by Peruvian Indians in the 17th Century, who employed crude extracts from the Cinchona trees to cure malarial fevers. These are but only a few of what modern medicine owe to ethnobotanical treasures.

There are four basic ways in which plants that are used by tribal peoples are valuable to modern medicine. First, some plants from the tropics are used as sources of direct therapeutic agents. For example, the alkaloid D - tubocurarine, extracted from a liane, Chondradendron tomentosum, is widely used as a muscle relaxant in surgery.

Secondly, tropical plants provide sources of starting points for the elaboration of more complex semi-synthetic compounds. For example, saponin, an extract from Dioscorea, is chemically altered to produce sapogenins, necessary for the manufacture of steroidal drugs. Thirdly tropical flora can serve as sources of substances that can be used as models for new synthetic compounds. Cocaine from the plant Erythroxylum coca, has served as a model for the synthesis of a number of local anesthetics, such as procaine. Lastly, plants can also be used as taxonomic markers for the discovery of new compounds. For example, although little was known of the chemistry of the Orchidaceae, plants of this family were investigated because of its close systematic relationship to the Liliaceae. The research demonstrated that not only was the Orchidaceae rich in alkaloids, but many of these alkaloids were unique and thought to be of extreme interest for the future. This rich natural economic resource needs urgent appraisal to coincide with the current "green wave" of lay interest in herbs and natural plant medicines, which is unparalleled in modern history.

We must consider seriously the importance of medicinal plants in the developing countries. The World Health Organization estimated that 80% of the Third World population rely on traditional medicine for primary health care needs. In many cases, these countries simply cannot afford to spend millions of dollars on imported medicines which they could produce or extract from their tropical forest plants. Indigenous medicines are relatively inexpensive; they are locally available and are usually readily accepted by the people. The ideal situation would be the establishment of local pharmaceutical firms that would create jobs, reduce unemployment, reduce import expenditures, generate foreign exchange, encourage documentation of traditional ethnomedical lore, and be based on the conservation and sustainable use of the tropical forests.

Conservation

What can the medical community do to aid both the struggle to conserve tropical forests and the search for new plant medicines? Many reasons have been presented to the general public: aesthetic, ethical and the like. But the most relevant to the medical profession is the utilitarian, that is, species are of direct benefit to us. The few examples that are given above (drugs from nature), are indicative of the kinds of undiscovered compounds that are undoubtedly there to be discovered.

Tropical forests are complex chemical storehouses that contain many undiscovered biomedical compounds with unrealized potential for use in modern medicine. We can gain access to these materials only if we study and conserve the plant species that contain them. The point that cannot be over-emphasized, and which is at the core of our argument here, is that biotic impoverishment is tantamount to chemical impoverishment. Loss of a species means loss of chemicals of possible use, chemicals potentially unique in nature, not likely to be invented independently in the laboratory.

Clearly, the most urgent conservation problems are occurring in the tropics. While the tropical forests cover less than 10% of the earth's surface, they are believed to contain over 50% of the world's species, and the majority of the endangered species. Extinction is a natural process, yet to view these recent extinctions as natural, is to misinterpret the geological record.

Parallel to this, is the urgent need to document and conserve ethnomedical plant lore, since indigenous knowledge is essential for use, identification and cataloguing of the (tropical) biota. As tribal groups disappear, their knowledge vanishes with them. Thus, the preservation of these groups is not a luxury, but a significant economic opportunity for the developing countries. Failure to document, this lore would represent a tremendous economic and scientific loss to humanity.

Action plan

To achieve these objectives ultimately, some practical issues need to be addressed to. These include:

(a) Formulating clear policies on the practice of traditional medicine. The policies should promote, inter alia, the organization of traditional healers, and realistic integration of traditional and modern medical practices.

(b) Promoting the volarization of medicinal and aromatic plants growing in the spontaneous flora, by setting up specialized units in agrobiological, pharmaceutical industrial and quality control aspects.

(c) Promoting the strengthening of research capability in the field of traditional medicinal plants.

(d) Promoting research in the economic mapping of the indigenous vascular plant flora, to identify the qualitative and quantitative natural resources, in medicinal and aromatic plants, in order to render the economic potential profitable.

(e) Promoting ethnobotanical studies to retrieve the vanishing ethnomedicinal information from remote village communities especially the traditional healers.

(f) Promoting the conservation of medicinal and aromatic plants, based on realistic in situ and ex-situ sustainable programmes, i.e., conservation of maximum plant biodiversity and individual plant species, respectively.

(g) Promoting meaningful infra-regional, regional and international cooperation that will enhance the exchange of information and technology of medicinal and aromatic plant genetic resources, without jeopardizing the genetic germ plasm.

References

Earthscan, J. (1982). What's Wildlife worth? International Institute for Environment and Development. London.

Eisner, T. (1988). Chemical Exploration of nature: A Proposal for Action, in Ecology, Economics, and Ethics: The Broken Circle. Yale University Press.

Farnsworth N.R. (1977). Foreword in major medicinal plants. J. Morton and G.C. Thomas. Springfied.

Plotkin, M.J. (1988). Conservation, Ethnobotany, and the Search for New Jungle Medicines: Pharmacognosy Comes of Age Again. Pharmacothera 8:257-262.

Sohultes, R.E. (1979). The Amazonia as a Source of New Economic Plants, Econ. Bot. 33: 259-266.

Swain, T. (1975). Plants in the Development of Modern Medicine.

Tyler, V.E. (1986). Plant Drugs in the Twenty-first Century. Econ. Bot. 40: 279 - 288.

UNESCO. (1978). Tropical Forest Ecosystems: A state of knowledge. Report prepared by UNESCO/UNDP/FAO. Paris.

Wagner, H. and Wolf, P., (1977). New Natural Products and Plant Drugs with Pharmaceutical, Biological and Therapeutic Activity. Springer-verlag. Berlin, New York.

Biotransformation of hydroxyanthraquinone glycosides in Cassia species

S.R. MALELE

Department of Pharmaceutical Sciences
Muhimbili Medical Centre
P.O. Box 65013
Dar es Salaam, Tanzania.

ABSTRACT

The development and application of tissue cultures in the production, biosynthesis and biotransformation of secondary metabolites is presented. Specific consideration is given to 1, 8 - dihydroxyanthraquinone derivatives of Cassia senna and Cassia artemisiodes. Plant Tissue Cultures, both static (solid) and in suspension (liquid) were established from seeds of same. Conditions for culture growth were investigated and optimised and cultures were maintained by sub-culturing for up to 32 passages.

Qualitative and quantitative analysis of hydroxyanthraquinone derivatives was investigated with emphasis on the application of HPLC. Total content and variation of these compounds in the species was carried out. Five compounds were identified and assayed, namely aloe-emodin, chrysophanol, emodin, physcion and rhein.

Incorporation of radio-active precursors (U-¹⁴C-acetate and (2-¹⁴C- malonate) were studied in cultures of the species, and their conversion into hydroxyanthraquinone derivatives has been instigated. Cultures were harvested at regular intervals, extracted and the hydroxyanthraquinones separated by HPLC before measurement of incorporated radioactivity.

Fluctuation of the radioactivity in the anthraquinone constituents occurred throughout the passage suggesting that biosynthesis and biotransformation were occurring simultaneously.

Plants of the same species were injected with (2-¹⁴C)-malonate, anthraquinones extracted at regular intervals and separated by HPLC prior to measurement of radioactivity.

Introduction

Anthraquinones are the largest group of natural quinones and historically the most important which for a long time have been used as dyes. The derivatives have cathartic activity and are used as purgatives and are widely employed in geriatric and pediatric medicine (Rada et al., 1974). Plant families which are the richest sources of this class of compounds (including important genera) are Polygonaceae (Rheum, Rumex and Polygonum), Rhamnaceae (Rhamaus and Zizyphus), Leguminoceae (Cassia), Rubiaceae (Morinda, Rubia and Galium, and Liliaceae (Threase and Evans, 1983).

Species such as Rheum palmatum (rhubarb), Aloe ferox, Cassia senna, and Rhamnus alnus have long been used as laxative drugs. They contain the anthraquinone derivatives, mainly as glycosides, which on hydrolysis yield aglycones which are hydroxyanthraquinone derivatives. The common polyhydroxyanthraquinone derivatives present in laxative drugs are 1,8 - dihydroxyanthraquinones (1,8 - DHAQ) and typical structures are given in Figure 1.

Fig. 1: Typical polyhydroxyanthraquinones

	R1	R2
Chrysophanol	Me	H
Emodin	Me	OH
Physcion	Me	OMe
Aloe-emodin	EtOH	H
Rhein	CO2H	H

Biosynthesis of anthraquinones

Leristner et al., (1969) and Fairbairn et al. (1972) established that naturally occurring anthraquinones are synthesized by two completely separate pathways. Thus those of the emodin type (with substituents in both terminal rings A and C) are usually derived through the acetatemalonate (polyketide) pathway in both higher and lower plants, while the alizarin (without substituents in ring A) type of anthraquinones are derived through the shikimic acid pathway.

Pharmacology and mode of action

Sennosides have the highest purgative activity, followed by rhein monoglcosides, whereas the anthraquinone glycosides are less active and the aglycones have the least activity (Fairbairn et al., 1949, 1965, 1970).

The mechanism of action of anthraquinone glycosides involves the systematic deposition of these compounds to the site of action in the intestine, enzymatic cleavage of the sugar groups and the slow oxidation of the resulting compounds, thus releasing the free anthraquinones which act on the intestines to produce peristalsis (Fairbairn, 1964).

Plant tissue culture

Over the centuries, plants have made a major contribution to the health of mankind, particularly through their use as spices, flavours, fragrances, vegetable oils, soaps, natural gums, resins, drugs, insecticides and other significant industrial, medicinal and agricultural raw materials. Scraag (1986) noted that despite substantial advances in microbial and chemical production methods, plants still remain the source of active ingredients of some 25% of prescribed medicines. The continued demand of these compounds has encouraged scientists to search for reliable alternative sources. One of the significant contributions to the manipulative powers of modern biologists has been the development of tissue culture techniques. Plant cells in culture have been expected to produce secondary metabolites which are characteristic of the whole plant (Rai, 1976). Several patents dealing with the production from cultures of metabolites such as allergens, dios-genin, L-dopa, ginsenosides, glycyrrhixin, etc have been registered (Staba, 1982; Bajaj, 1988).

In this paper the establishment of tissue cultures of Cassia species and the careful phytochemical investigation of the controlled production of the hydroxyanthracene derivatives is discussed. An attempt to devise a sensitive, rapid and efficient analytical technique of these very closely related hydroxyanthracene derivatives by the use of HPLC will also be presented.

Materials and methods

Cultures

Cultures were established from seeds of Cassia artemisioides on Murashige and Skoog's modified tobacco medium. Cultures were incubated in the dark at 25°-27°C and maintained for more than 30 passages, each of 38 days. Static cultures were chosen for subsequent analysis rather than suspension cultures because they proved to give better results in the production of secondary metabolites. Anthraquinone content variation during a single passage of the culture was done with a view to subsequent investigation of the biotransformation of the compounds produced.

Phytochemical investigations

The phytochemical investigations followed the scheme shown in Figure 2.

Sensitivity screening for sennosides showed negative results. Nonetheless purification was carried out by column chromatography and preparative TLC. Five compounds - chrysophanol, emodin, physcion, aloe-emodin and rhein -were isolated and identified spectroscopically (UV, IR and MS) and by comparison of the melting points with those reported for chrysophanol, emodin, physcion, aloe-emodin and rhein.

Radio-tracer studies

Feeding technique

The precursors used were (1-¹⁴C)-acetate and (2-¹⁴C)-malonate. 0.1 mCi in 5 ml of each of the tracers was separately added onto the callus once the culture showed visible signs of growth. Cells were harvested at regular intervals, extracted and the compounds were separated by high performance liquid chromatography (HPLC). Plants were fed with ¹⁴C-malonate and radio-active incorporation monitored at regular intervals by HPLC. The malonate was fed at the leaf-base where an axillary bud was evident. The HPLC instrument consisted of Rheodyne rotary valve which was equipped with a 100 ml loop, in order to collect sufficient eluate from the column for scintillation studies.

Anthraquinones were consistently eluted in the sequence, aloe-emodin, rhein, emodin, chrysophanol and physcion. Using the reverse phase system, this elution sequence is broadly in accordance with their polarities: aloe-emodin polar, and physcion, least polar is eluted last.

Results

The results of the study to investigate the influence of ¹⁴C-acetate and ¹⁴C-malonate, intermediates in the biosynthesis of polyketides, on the production of hydroxyanthracene derivatives are shown in Figure 3A and 3B and also in Figure 4A and 4B. The incorporation rates of the two radio-tracers and the radio-activity values are given in Table 1.

Figure 2 : Schematic diagram for the entraction of Hydroxyanthracene derivatives

Figure 3A : Influence of acetate and malenate in rhein production in static cultures of Cassia senna

Figure 3B : Influence and radioactivity incorporation of acetate and malonate in emodin in static cultures of Cassia senna

Figure 4A : Influence and radioactivity incorporation of acetate and malonate in chrysophanol in static cultures of Cassia senna

Figure 4B : Influence and radioactivity incorporation of acetate and malonate in aloe-emodin in static cultures of Cassia senna

Fig 5 : Suggested transformation of anthroquinones derivatives

Comments:

(a) Anthracene derivatives were able to absorb the radio- tracers.

(b) Malonate was incorporated into hydroxyanthracene compounds at a higher rate than for acetate. The incorporation varied, chrysophanol being highest and with rhein much lower.

The suggested transformation of anthraquinone derivatives is given in Figure 5.

Discussion and Conclusion

From the results above the interconversions shown in Scheme 1 were found to occur.

Scheme 1

Le médicament indigène Africaine: Sa standardisation et son évaluation dans le cadre de la politique des soins de santé primaires

MAMADOU KOUMARE

WHO Africa Office
Brazaville, Republic of Congo

Sommaire

Le médicament indigène africain obéit à des règles de préparation dont le respect permet d’obtenir des produits d’une standardisation acceptable, qualitativement et quantitativement.

L’étude des doses thérapeutiques proposées par le tradithérapeute, montre que ces doses sont également acceptables.

L’efficacité thérapeutique étudiée par des essais cliniques comparés et l’importance de la consommation du remède indigène africain, constituent les éléments de son évaluation. Les règles de cette évaluation devrait tenir compte du concept du médicament indigène africain.

Introduction

Il ne fait plus aujourd’hui aucun doute que les soins de santé primaires (SSP) offrent l’une des approches les plus viables pour atteindre l’accessibilité à la santé pour tous.

En effet, cette approche suppose la prise en compte de toutes les ressources appropriées disponibles y compris les pratiques et les remèdes des systèmes indigènes de soins.

La composante pharmaceutique de cette politique des soins de santé primaires, de mande la mise à la portée des populations, géographiquement et économiquement, des médicaments appropriés.

Malgré l’engouement populaire, l’acceptabilité du médicament indigène africain se heurte aujourd’hui encore à une certaine méfiance; d’où la nécessité de son évaluation et de sa standardisation afin d’en favoriser son homologation et son inscription sur les listes des médicaments essentiels.

Si tout le monde est unanime sur la nécessité d’une évaluation, il ne semble pas qu’il en soit de même pour le recours aux conditions de mise sur le marché appliquées actuelle ment aux nouveaux médicaments.

Notre propos n’est point de faire accepter n’importe quel médicament pour les soins de santé primaires, ni encore moins d’opposer le remède indigène africain au médicament européen; mais de présenter une expérience ayant pour objectif de dissiper la méfiance causée par certains préjugés défavorables et d’aider à résoudre le problème de santé publique qu’est l’approvisionnement régulier des formations sanitaires en médicaments.

Si une certaine analogie est apparente dans le concept de médicament des deux systèmes de soins, indigène africain et exotique européen, il n’en est pas moins vrai que la philosophie qui les soutend est différente: l’un relève de l’esprit analytique, du raisonnement et de l’expérimentation; et l’autre, de l’esprit systémique, de l’intuition et de l’empirisme.

Au prime abord on pourrait penser que les médicaments du système exotique européen traitent les causes de la maladie et que ceux du système indigène africain soignent les symptomes.

Nous nous empressons d’ajouter qu’il ne serait pas juste de dire que les médicaments indigènes africains ne sont utilisés que pour des traitements symptomatiques.

Comme nous l’avons déjà dit et écrit, chacun des deux systèmes de soins dispose de “médicaments étiologiques” et de “médicaments symptomatiques” dont l’élaboration répond à certaines règles. Il nous à semblé indispensable et urgent de faire le point sur ces règles afin de définir leurs limites de fiabilité et de permettre une meilleure standardisation du remède indigène africain.

Pour ce faire, nous avons essayé de suivre le processus de son élaboration et de son administration.

Elaboration du médicament indigène Africain

La méfiance dont nous avons parlé plus haut, pour ne pas dire la crainte, persiste encore vis-à-vis du remède indigène africain malgré l’engouement des populations.

On ne peut nier qu’elle soit justifiée; mais malheureusement, on accuse trop souvent et abusivement la qualité ou les doses thérapeutiques du médicament indigène.

“Les faux guérisseurs” sont hélas trop nombreux et il n’est point question de vouloir garantir leurs préparations et leur compétence.

Loin de nous l’idée de nier les insuffisances de l’art pharmaceutique traditionnel africain; mais il nous parait injuste de ne pas reconnaître qu’il existe des règles de préparation et d’administration bien adaptées au système. Il suffit pour s’en convaincre, de savoir que dans certains pays on à déjà procédé à la codification des règles de la médecine indigène en général et du remède indigène en particulier.

Matières premières

Nous nous limiterons volontairement aux plantés médicinales qui constituent actuellement la majeure partie de ces matières premières et dont les techniques de récolte nous paraissent les plus respectées pour ne pas dire les mieux standardisées.

Le respect scrupuleux des règles de récolte trouve son explication dans les craintes que le phytothérapeute éprouve dans leur transgression. Le geste qui semble le plus anodin n’est pas négligé; et nous ne sommes pas de l’avis de ceux qui ne voient toujours dans leur exécution que superstition. Et même si une possible superstition il y avait, il serait souhaitable de ne pas la combattre avant d’en connaître l’origine ou d’en faire son évaluation complète; il y va de la préservation de bonnes conditions de remassage et de l’obtention d’échantillons moyens de matières premières faciles à tester et à homologuer, à ce point de vue, la présentation sous forme de bottes à retenu notre attention; et nous avons essayé d’en connaître le poids approximatif par espèces végétales.

Cette homologation, d'après notre modeste expérience, est beaucoup plus aisée au niveau des phytothérapeutes qu’au niveau des herboristes soucieux surtout de la vente de leurs produits malgré l’homogénéité apparente des bottes.

L’identification de la plante n’est pas seulement morphologique; c’est un véritable diagnose que pratique le phytothérapeute à partir également des caractères Il connaît en outre la période et le lieu de récolte, la partie de la plante qui lui permettent d’assurer des succès constants. Malheureusement beaucoup d’enquêteurs ne s’en préoccupent pas sur le terrain et ne posent pas suffisamment de questions. Il est rare que le phytothérapeute conserve les matières premières au delà d’une année; ce qui n’est point le cas chez les herboristes.

A notre avis, avec l’identification faite par le phytothérapeute, la connaissance de la partie utilisée de la plante, des techniques, période et lieu favourable à la récolte, il est possible d’établir les bases d’une homologation acceptable à partir d’échantillons moyens.

Il est certain, qu’il foudra ensuite que les institutions chargées de l’étude des plantes médicinales améliorent progressivement cette connaissance en la complétant par d’autres caractéristiques que ne peuvent apprécier les tradipraticiens de santé. C’est la méthode d’approche qui nous à conduit à déterminer la bonne période de récolte, les grands groupes chimiques, les tenues en eau, cendres totales, huiles essentielles, etc.. Si l’éthique médicale traditionnelle oblige le phytothérapeute au respect rigoureux de règles définies de récolte, elle conseille au contraire une adaptation de la préparation et du traitement au patient. Cette pratique rend plus difficile une standardisation dans le cadre d’une fabrication industrielle du médicament.

Composition du médicament

La standardisation qualitative et quantitative de la composition du médicament indigène africain s’avère une nécessité dès lors que sa fabrication industrielle ou même semi-industrielle est envisagée; car les règles des préparations individuelles que peuvent préconiser les tradithérapeutes deviennet difficilement applicables. Il est cependant indispensable de ne pas trop s’en écarter sans analyse critique préalable comme nous l’avons déjà préconisé pour les techniques de récolte.

Sur le plan qualitatif, il n’est point aberrant de constater que certains médicaments indigènes africains contiennent plus de dix constituants. Le seul terme “excipient” de certains médicaments qui sont inscrits dans un répertoire sérieux comme le “vidal” peut en contenir autant. C’est pourquoi, il est indispensable comme nous l’avons suggéré, de ne rien considérer à priori comme inutile. On pourrait cependant, à la lumière des entretiens avec le tradithérapeute et de certains essais chimiques, pharmacologiques et/ou cliniques, éliminer certaines drogues qui ne modifient pas notablement ni l’acceptabilité, ni la stabilité, ni l’innocuité et l’efficacité du médicament. Telle est notre méthode d’approche.

Concervant les quantités, il suffit de se les faire indiquer par le tradithérapeute, de procéder aux mesures pondérales ou volumétriques appropriées de chaque constituant; celles-ci pouvant ultérieurement être reproduites facilement à partir des moyennes établies après plusieurs mesures.

Pour faciliter les modes de préparation, nous avons commencé par adopté le matériel de cousine qu’utilise le tradithérapeute; puis, au fur et à mesure qu’on établissait les valeurs limites de certains caractères, on remplaçait ce matériel de cuisine par un appareil de pharmacotechnie appropriée; ainsi on finit par établir une certaine équivalence entre les deux outils de travail et favoriser les échanges et le dialogue entre les systèmes de soins.

Formes médicamenteuses et modes d’obtention

S’il nous à été relativement facile d’établir des équivalences entre la ténuité des poudres obtenues à partir des tamis locaux de cuisine et ceux de notre broyeur Forplex, il n’en à pas été de même pour les autres formes galéniques. On peut cependant affirmer que l’observation rigoureuse des modes opératoires permet de garantir, dans une certaine mesure, la reproductibilité des caractéristiques des préparations et par voie de conséquence, celle des doses.

C’est ainsi que pour une décoction par exemple le tradithérapeute tiendra compte à la fois:

Sur le plan qualitatif de:

(i) la couleur du décocté;
(ii) la viscosité, le cas échéant;
(iii) le goût (astringence);

Sur le plan quantitatif:

(i) du nombre de bottes de plantes;
(ii) des volumes d’eau au début et à la fin de l'opération, souvent identiques respectivement par l’immertion et la non-immretion des bottes de plantes et non par le temps d’ébullition.

Le contrôle de cette reproductibilité peut se faire sur l’extrait sec obtenu à partir du décocté en définissant qualitativement et quantitativement certaines propriétés et caractéristiques.

Concernant une des critiques les plus fréquentes, celle des conditions hygiéniques de préparation, là aussi nous pensons qu’il n’est pas juste de dire que le tradithérapeute n’en à aucun souci. Les techniques de filtration ou de décantation et l’usage des récipients neufs n’ayant pas encore servi auxquels il à recours, de même que la prise en compte des formes pharmaceutiques (surtout le décocté; donc après ébullition) et de la voie d’administration (surtout orale ou externe) expliquent en partie la situation.

Nous livrons pour réflexion un des principes fondamentaux de la médecine indigène africaine:

l’organisme humain à besoin d’un équilibre symbiotique et ne pourrait subsister dur une stérilité absolue.

Administration du médicament indigène Africain: la posologie

L’existence des doses dans la médication traditionnelle africaine à été souvent contestée, à notre avis, on à parfois imputé à tort à cette médication des accidents causés par l’imprudence des victimes elles-mêmes. Notre propos est beaucoup plus d’affirmer l’existence de doses thérapeutiques acceptables que de nier l’insuffisance de la précision des unités de mesures.

Faire mieux connaître les règles qui régissent la détermination des doses afin d’en permettre son amélioration est l’un de nos objectifs.

Comme nous l’avons dit, le respect rigoureux des modes de préparation permet d’obtenir des médicaments comparables dans des limites qu’apprécie valablement le tradithérapeute et qu’une institution sommairement équipée peut déterminer d’une manière plus précise. Pour ce faire, sans nous préoccuper du principe actif, nous cherchons à suivre qualitativement et quantitativement certains constituants (au moins deux) et certaines caractéristiques (physico-chimies et/ou organoleptiques) qui nous permettent d’attester que les préparations sont comparables. L’existence des formes pharmaceutiques non unitaires nécessite la connaissance des règles de mesures des prises avec les moyens utilisés à cet effet.

Il ne suffit pas par exemple d’utiliser la même cuillère et le même produit pour croire que les quantités de poudre mesurées sont égales. En effet, pour avoir la même quantité il faut obligatoirement respecter la règle de la mesure rase.

Par ailleurs, l’utilisation de la cuillère demande qu’on précise s’il s’agit de la cuillère à café, & dessert ou à soupe.

De même, en pratique traditionnelle, il faut savoir que la pincée s’effectue verticalement et est limitée à la première phalange; qu’il faut bien préciser le nombre de doigts, à défaut duquel on retient la pincée à deux doigts.

L’étude pondérale des bottes de plantes fraîches nous à donné une variation du simple au triple (1,3 à 3,1). Celle des pincées une variation de 1 à 2,5 (voir annexes).

Par voie orale, les quantités de décocté absorbées par les malades sont fonction de la capacité de leur estomac dont les limites de variation (1 litre à 1,5 litre pour l’adulte) permettent aux tradithérapeutes de préconiser comme il le font, la boisson de certaines tisanes.

En prenant encore l’exemple du répertoire Vidal, on constate que la dose usuelle journalière chez l’adulte peut varier souvent de 1 à 3 comprimés; autrement dit, du simple au triple.

La comparaison entre ces différents chiffres nous permet de dire à notre avis que les variations de doses thérapeutiques préconisées par le tradithérapeute sont acceptables.

Nous pensons que la détermination de la dose à administrer dépend aussi de la compétence du praticien; et cela est valable pour les deux systèmes de médecine.

C’est au médecin d’adapter cette dose usuelle journalière aux différents cas. Seule son expérience lui permettra d’éviter les erreurs d’appréciations et les accidents. L’attitude du tradithérapeute comme celle du médecin sera dictée par l’état général du malade, de son sexe, de son âge, de sa corpulence (pour le tradithérapeute surtout) ou de son poids (pour le mèdicin) et de la gravité de son mal.

Evaluation du médicament indigène Africain

Eléments d’évaluation L’efficacité thérapeutique et l’importance de l’usage du médicament indigène africain constituent sans nulle doute des éléments de son évaluation.

· En effet, il n’est point besoin de rappeler ici les bons résultats de certaines préparations traditionnelles qui sont à la base de la découverte de produits purs cristallisés et de la synthèse de substances analogues.

· La popularité recueillie pendant des décennies “(pharmacovigilance estimative)” et la grande consommation d’un médicament indigène permettent de situer son importance dans la couverture des besoins pharmaceutiques et juger de l’opportunité de son inscription sur la liste des médicaments essentiels.

La méthode d’évaluation à notre avis, devrait être la comparaison (par essais cliniques) avec un médicament déjà existant sur le marché et jouissant d’une très bonne acceptabilité aussi bien sur le plan de coût que sur le plan d’efficacité et de disponibilité.

Conditions préalables de l’évaluation du médicament indigène africain

La mue sur le marché d’un médicament obéit aujourd’hui à des conditions de rigueur qui, si elles sont nécessaires et indispensables pour les nouvelles molécules, ne nous paraissent pas justifiées pour le médicament indigène ayant subi et vaincu l’épreuve du temps après administration à l'espèce humaine. Ceci signifie en effet que la pharmacovigilance, autrement dit la surveillance des effets des médicaments dans leurs conditions usuelles d’emploi ne lui à pas été défavorable.

Loin de nous l’idée de nier toute possible toxicité tératogène de ces remèdes; mais nous pensons également qu’il n’est pas juste de minimiser le fait qu’ils ont vaincu l’épreuve du temps après administration à l’homme et non à un animal de laboratoire. C’est pourquoi, nous préconisons une adaptation des conditions administratives et législatives, de mue sur le marché afin quelles soient appropriées et favorisent l’innovation au lieu de la freiner.

C’est ainsi que nous pensons que cette adaptation doit se faire en autorisant les es sais cliniques comparés plus rapidement qu’ils ne le sont actuellement; tout au moins légalement et officiellement.

Le problème posé est plus éthique que scientifique; c’est pourquoi la solution doit être conforme à l’éthique de notre environment socio-culturel.

Conclusion

Au terme de cette communication, nous pensons avoir exposé avec assez de clarté notre méthode d’approche, nos résultats et nos conclusions en ce qui concerne la standardisation et l’évaluation du médicament indigène africain.

Nous nous sommes à comprendre les attitudes et concepts qui sont à la base des insuffisances des pratiques afin de trouver les moyens de les rendre reproductibles.

Nous tenons à ajouter que cette approche ne s’oppose nullement à la prise en compte ultérieure d’études plus approfondies sur par exemple, s’il existe, le principe actif, sa toxicité et son mécanisme d’action.

Sans nier leur importance, notre priorité n’est point de rechercher un principe actif; de déterminer une DL 50, ou un mécanisme d’action; mais plutôt de s’assurer de la reproductibilité et de la stabilité des préparations avec des normes de spécifications; car il s’agit là de médicaments pour lesquelles l’épreuve de la pharmacovigilance n’a pas été défavorable.

Pour ce faire, nous pensons que la constitution d’échantillons moyens sur une période donnée de récolte et le respect rigoureux de certaines règles suffisent.

Le “remède indigène amélioré”, comme nous l’avons appelé, peut, à la faveur d’une adaptation des conditions de mise sur le marché conforme à l’éthique de notre environnement socio-culturel, être accepté et produit au moins semi-industriellement afin de répondre dans l'immédiat au problème de la santé publique qu’est l’approvisionnement en médicaments des formations sanitaires.

Bibliographie

Delmas, À. (1970). Anatomie humaine, descriptive et topographique. Ed. Masson Paris.

Kayser, C. (1963). Physiologie: Fonctions de Nutrition. Ed. Flammarion Paris.

Koumare, M. (1978m). Le Remède traditionnel africain et son Evaluation. Bulletin Sante pour Tous, 3: 28-33, Bamako

APPENDICES

1. Evaluation des Bottes de Plantes Fraîches (en g)

No. d’ordre	Guiera senegalensis	Diospyros mespiliformis	Saba senegalensis	Opilia celtidifolia	Bridelia ferruginea (saguan)	Parkia biglobosa (nere)
1	110,2	185,5	182	51,5	232,2	181,5
2	140,4	191,8	177,5	130,2	226,1	220,9
3	116,1	224,4	166,4	164	257,2	169,8
4	122,8	149,4	190,9	142,7	194,9	252
5	161,9	184,3	155,1	105,8	206,4	184,2
6	130,7	230,3	138,8	115,8	184	179,2
7	167,6	191,8	177,5	130,4	190,6	136,5
8	113,5	212,5	113,2	140,2	215,7	153,2
9	147,9	212,3	192	136,5	187,6	104,7
10	122	207,9	189,8	94,6	194,1	193,3
11	1333,1	1990,2	1683,2	1211,7	2088,8	1775,3
Average	133,31	199,02	168,32	121,17	208,88	177,53

2. Calcul des Variations des Mesures de Pincées de la Poudre D’asthmagardenia

Désignation des séries de mesures	Mesure extra inférieure (Mi)	Mesure extrème supérieure (Ms)	Report Ms Mi
1	0,2073	0,5278	2,5
2	0,1976	0,3212	1,6
3	0,1966	0,3282	1,6
4	0,2310	0,3443	1,5
5	0,2542	0,3600	1,4

Bottes de Plantes Fraîches

Désignation des plantes	Mesure extrême Inférieure (mi)	Mesure extrême supérieure (ms)	Rapport Ms
Guiera senegalensis	110,2	167,6	1,5
Diospyros mespiliformis	149,4	230	31,5
Saba senegalensis	113,2	192	1,6
Opilia celtidifolia	51,5	164	3,1
Bridelia ferruginea	184	257	21,3
Parkia biglobosa	104,7	252	2,4

D’asthmagardenia

No. d’odre	Tare lare + Poudre		Poudre(g)
1	5,9558	6,2604	0,3046
2	6,2375	6,5157	0,2782
3	6,4909	6,6982	0,2073
4	5,8706	6,3035	0,4329
5	6,3572	6,7570	0,3998
6	5,7518	6,2796	0,5278
7	6,3614	6,7975	0,4361
8	6,1505	6,5910	0,4405
9	6,1310	6,4805	0,3495
10	6,0659	6,4950	0,4291

My = 0,3805 gm

D’asthmagardenia

No. d’odre	Tare Tare + Poudre		Poudre(g)
1	6,2980	6,5042	0,2062
2	6,1622	6,4777	0,3155
3	6,7003	6,9084	0,2081
4	6,2670	6,4646	0,1976
5	6,6130	6,9342	0,3212
6	6,1116	6,3390	0,2274
7	6,6522	6,9386	0,2864
8	6,2492	6,5276	0,2784
9	6,2055	6,4426	0,2371
10	5,6594	5,9298	0,2704

My 0,2548 g

6. Evaluation de la Pincée de la Poudre

D’asthmagardenia

No. d’odre	Tare Tare + Poudre		Poudre (g)
1	6,2980	6,5032	0,2052
2	6,1620	6,3862	0,2242
3	6,7005	7,0287	0,3282
4	6,2672	6,5349	0,2677
5	6,6130	6,8559	0,2429
6	6,1113	6,3628	0,2515
7	6,6522	6,8795	0,2273
8	6,2489	6,4455	0,1966
9	6,2058	6,4220	0,2162
10	5,6593	5,8643	0,2050

My = 0,2364 g

7. Evaluation de la Pincée de la Poudre D'asthmagardenia

No. d’odre	Tare Tare + Poudre		Poudre (g)
1	6,5,9559	6,2130	0,2571
2	6,2381	6,5824	0,3443
3	6,4914	6,7793	0,2879
4	5,8704	6,1430	0,2726
5	6,3573	6,6548	0,2975
6	5,7522	6,0433	0,2911
7	6,3616	6,6746	0,3130
8	6,1508	6,3818	0,2310
9	6,1312	6,4310	0,2998
10	6,0663	6,3322	0,2659

My = 2860 g

8. Evaluation de la Pincée de la Poudre

D’asthmagardenia

No. d’odre	Tare Tare + Poudre		Poudre (g)
1	8,8108	9,1197	0,3089
2	6,1620	6,4421	0,2081
3	6,7005	6,9547	0,2542
4	6,2673	6,5570	0,2897
5	6,6130	6,9702	0,3572
6	6,1112	6,4504	0,3392
7	6,6524	6,0124	0,3600
8	6,2493	6,5490	0,2997
9	6,2057	6,5402	0,3345
10	5,6595	5,9712	0,3117

My = 3135 g

Chemical Evaluation of Tanzanian medicinal plants for the active constituents as a basis for the medicinal usefulness of the plants

MAYUNGA H. H. NKUNYA*, H. WEENEN**, & D. H. BRAY***

*Department of Chemistry, University of Dar es Salaam P. O. Box 36061, Dar es Salaam, Tanzania

** Quest International, P. O. Box 2, 1400 CA Bussum, The Netherlands.

***London School of Hygiene and Tropical Medicine Keppel Street, London WC 1E 7HT, U.K.

ABSTRACT

Drugs derived from medicinal plants still form the basis for rural medical care in most developing countries, apparently either because of lack of modern medical facilities in these areas, or as a supplement to the latter. In practice, most of these drugs offer effective treatment. This is not surprising because about 40% of all pharmaceutical presently in use are derived from natural sources (plants, fungi and other microorganisms, animals, etc.), either used directly as such, or with some modifications. Unfortunately, the we of crude plant extracts without any scientific evaluation, could lead to serious complications. Ineffective drugs could be used just as a matter of belief or tradition; under/over-doses could be taken; highly toxic drugs with short term, long term, or cumulative effects could be prescribed etc. The last two effects, however, are much more difficult to recognise than the others, and hence potentially more serious. In addition to these, the preparation, handling and storage of the drugs could lead to decomposition or transformation of the hitherto active constituents to ineffective and/or harmful products. Thus there is a need to evaluate and establish a scientific rationale for the use of the traditional medicinal plants, through chemical, pharmacological, toxicological and microbiological studies. In this paper, chemical investigations of medicinal plants for the active constituents and the correlation between biological activity of the crude extracts and/or the pure chemical constituents with the medicinal uses of the plants will be discussed.

Introduction

Quite a number of plants are used in different parts of the world for the treatment of various ailments. The medicinal values of most of these plants were recognised since ancient times. In fact, it can correctly be argued that the development of modern pharmaceutical is based on this ancient knowledge of medicinal plants and traditional medicines. Thus presently, about 40% of pharmaceuticals are derived from natural sources (plants, microorganisms, fungi and animals (Farnsworth, 1984). These drugs are used as such, or as derivatives. Moreover, several natural products obtained from medicinal plants, which cannot hitherto be used as such, have offered leads to the development of various pharmaceuticals, as analogues or derivatives.

In developing countries, traditional medicines from plants continue to form the basis of rural medical care. This is so because, obviously, these medicines are easily available and cheap. However, the use of such medicines in their crude forms without establishing scientifically their efficacy and safety could, in a short while or long run, be detrimental to the very health of mankind. Therefore, there is an urgent need to carry out scientific evaluations of these medicines worldwide. After all, apart from the efficacy and safety of traditional medicines, the scientific evaluation may lead to the isolation of a pure active ingredient which otherwise occurs, in minute quantities in the crude drug. And since medicinal plants depend on their geographical location, such isolated active principle can then be synthesized cheaply, so that eventually the drug is available to a larger population. Alternatively, knowledge of the structures of naturally occurring, medicinally useful compounds may give leads to the synthesis of analogues, which could be cheaper, and sometimes even more active than the naturally occurring compounds.

In 1976 we initiated a long term project on the scientific evaluation of Tanzanian medicinal plants, aimed at establishing the active constituents. So far we have studied plants which are used for the treatment of bacterial and fungal diseases (Sawhney et al., 1978a and 1978b; Khan et al., 1980), and those which are used for malaria. Occasionally we also evaluated the isolated compounds for antitumour or other activities. In this paper results of our on-going research on plants used in Tanzania for the treatment of malaria and malaria-related fevers will be discussed. Prof. Khan will present our results on the chemical investigations of plants used for bacterial and fungal diseases (Khan and Nkunya, 1990).

The malaria problem

Malaria is one of the most prevalent tropical and subtropical diseases (WHO, 1982/83). Recently it has been estimated that about 260 million people are infested annually (WHO, 1988). In tropical Africa alone about one million children under 14 years die from the disease annually (Underson, 1986). It is now over forty years since campaigns to eradicate the disease were initiated but, unfortunately, until now there is no success in eradicating this disease in the poor, developing countries. Efforts to develop an antimalarial vaccine have been futile because of the complicated stages of malaria infestation (Mgani, 1990).

Efforts to eradicate the mosquito vector, the Anopheles mosquito, have been futile because of financial and management problems of the eradication programmes. Furthermore, the mosquitoes are now known to be developing resistance against the cheap insecticides, such as DDT, fenitrothin, proppoxur, malathion, clorfoxin, and synthetic pyrethrins, which are generally used in these programmes (WHO, 1984). The use of large quantities of these insecticides also poses an environmental problem, since some of them, such as DDT, are non-biodegradable. The economic difficulties being faced by the affected countries, coupled with the emergence of other killer diseases, such as AIDS, will, most likely, hamper financial commitments in the fight against malaria, particularly the massive mosquito eradication programmes, since these involve huge financial requirements.

Due to the above constraints, at the moment, malaria chemotherapy should be given due attention. But again sad news have emerged in this direction. That is, the most dangerous human malaria parasite, Plasmodium falciparum, is developing resistance against the commonly used cheap drugs such as quinine and chloroquine (Breman and Campbell, 1984). The use of the new drugs, mefloquine, fansidar, amodiaquine, primaquine, etc, in malaria chemotherapy, poses other problems. These drugs are quite expensive and some have serious side effects. They particularly affect human liver, kidneys and the nervous system (Mtulia, 1976). Hence, at present, chloroquine and quinine continue to be prescribed to malaria patients. Larger doses of chloroquine are now being recommended for drug resistant strains of P. falciparum. However, long-term effects of such large doses of chloroquine we still unknown, but could be significant.

Due to the shortcomings discussed above, efforts are now being directed in obtaining drugs which have structural features that are different from those of chloroquine and related drags, and those of sulfa drugs, either synthetically or from plants.

Antimalarials from plants

After the isolation of quinine from Cinchona trees (Sterling, 1977), and artemisinine from Artemisia annua L. (Compositae) (Xu-Ren et al., 1985), it has become apparent that plants are a potential source of antimalarial drugs. Artemisinine (also known as ginghaosu) is one of the most potent antimalarial drugs known at present, which is toxicologically the safest (Xu-Ren et al., 1985). Since this compound has a structural feature which is different from that of any other known antimalarial, parasite resistance to this compound is unlikely to take place in the near future.

The drug is still obtained from the plant where it occurs in small quantities, since its synthesis is still very cumbersome (Gavagan, 1988). This makes the drug to be very expensive. It is, therefore, worthwhile to put more efforts in searching for other potent and abundant antimalarials from medicinal plants, or other sources, while efficient and cheap synthetic methods for artemisinine and its derivatives are being developed. That is why at present enormous efforts are being exerted in searching for antimalarials from medicinal plants, and several leads have so far been obtained. Thus, the vascular plant famines Amaryllidaceae, Meliaceae, Rubiaceae and Simaroubaceae, have been found to include plant species which are active against malaria parasites (Spencer et al., 1947), and several active compounds have been isolated from some of these plants. Several quassinoids, which were isolated from some plants of the family Simaroubaceae, showed potent antimalarial activity in vitro (e.g., see WHO, 1984; Thaithong et al., 1983). The compounds also - owed a strong mammalian cytotoxicity. However, preliminary studies on the structure- activity relationship of quassinoids have shown that the structural requirements for antimalarial activity and cytotoxicity are different (e.g. see Bray et al., 1987). Therefore, one can expect that structural modifications of these compounds to suppress cytotoxicity, if feasible, can be performed to give modified compounds which might be safe antimalarials However, up to now such modifications have not been performed (Phillipson, 1990).

Recently, Prof. Hostettmann from Switzerland has found that the crude extract from Psorospermum febrifugum (Guttiferae) possesses an antimalarial activity at a level similar to that of artemisinine (Hostettmann, 1990). He has isolated the active constituents from the plant, and further evaluation of this compound for its potency as an antimalarial drug is in progress.

Antimalarials from Tanzanian medicinal plants

In our on-going research on Tanzania antimalarial plants, we have screened crude extracts from leaves, stem and root bark of sixty medicinal plants. The results are shown in Table 1 (Weenen et al., 1990). Some of the most active plants were the tubers of Cyperus rotundus L. (Cyperaceae), and the root bark of Hoslundia opposita Vahl. (Labiatae). Chemical studies of the C. rotundus extracts led to the isolation of a number of compounds, some of which were active against the multidrug resistant K1 strain of P. falciparum malarial parasite in vitro. These included a-cyperone (1) and (+)-b-selinene (2) (Weenen et al., 1990b). However, the activity of 2 appeared to be due to decomposition products. Thus, whereas the undercomposed compound was inactive, the decomposed material was active.

We have isolated three new compounds from the root bark of H. opposita which we have named hoslunone (3), hoslundione (4) and hoslundin A (5) (Marandu, 1990). All these compounds were active against P. falciparum malaria parasites in vitro. The crude H. opposita extract also gave several other active compounds, which were in minute quantities, and hence their structures could not be determined. We are now re-investigating the plant in order to obtain larger quantities of the compounds so that their structures can be identified.

Other active plants in our investigation were Margaritaria discoidea (Baill.) Webster (Euphorbiaceae), from which securinine (6) was obtained and found to be the active principle, and Zanthoxylum gilletii (De Wild) Waterm. (Rutaceae), which contains two active compounds, pellitorine (N- isobutyldec-2, 4-dienamide) (7), and fagaramide (8) (Weenen et al., 1990b). Another compound (9) was obtained from the latter plant as well, but this metabolite, despite its novel chemical structure, was inactive (Kinabo, 1990).

All the compounds 1, 3-7 shown above, contain an a,b-unsaturated carbonyl moiety. It is believed that their antimalarial activity is due to the ability of the nucleic acids of P. falciparum malaria parasites to react with the a,b-unsaturated carbonyl moiety, in a Michael addition fashion (Weenen et al., 1990).

We also isolated several compounds from the crude root bark extract of Artemisia afra Wild (Composite) (same genus as Artemisia annua, the source of artemisinine) but none of the isolated compounds had any marked activity (Kinabo, 1989).

Azidarachta indica A. Juss. (Mwarobaini in Swahili)

Azidarachta indica is widely used in East and West Africa for the treatment of malaria and malaria related fevers. We therefore included this plant in our investigations. Results on the antimalarial activity of this plant are given in Table 1 (Weenen et al., 1990a). As it can be noted, the plant showed only a mild activity. Apparently, the active component from this plant, which has recently been isolated in India, occurs in very minute quantities (Philipson, 1990). This might be the reason for the mild activity of the crude extract.

Antimalarials from plants of the genus Uvaria

Uvaria species have proved to be rich in a variety of compounds, some of which exhibit a wide range of biological properties, such as antibacterial, antifungal, and anticancer activities, and pharmacological properties (Leboef et al., 1982). The chemistry and biological activities of these compounds have attracted interests in investigating these plants phytochemically. That is why in the course of our investigations on antimalarial plants, we decided to screen the Uvaria species, which grow in Tanzania, for their antimalarial activity, and ultimately isolate the active principles and/or any other chemically interesting compounds. After all, most of these Uvaria species (commonly known is Mshofu or Msofu) are used for the treatment of malaria (Kokwaro, 1976).

We have screened nine Uvaria species which were collected from different parts of Tanzania. Their activities are summarised in Table 1 (Nkunya, et al., 1990). It can be noted from Table 1 that all nine plants are active against the multidrug resistant K1 strain of P. falciparum malarial parasite, leaf extracts being the least active. Table 1 also shows that most of the activity is concentrated in the less polar or medium polar compounds, which are soluble in petroleum ether or chloroform.

Several compounds have been isolated from the most active extracts, and these have been assayed for their activity against the multidrug resistant K1 strain of P. falciparum malaria parasites (Nkunya, et al., 1990a). C-Benzylated dihydrochalcones (the uvaretins) (Mgani, 1990, Nkunya, 1985), and sesquiterpeneindoles (Nkunya et al., 1987a, Nkunya and Weenen, 1989, Nkunya et al., 1990b) have been found to be the active components of these plants. The activity of the dihydrochalcones was found to depend on the presence of free hydroxyl groups, and on the molecular size of the compounds (Nkunya et al, 1990a). That is, small molecules showed a higher activity than large ones. The activity of the sesquiterpeneindoles appears to be due to the sesquiterpene side chain and not the indole moiety. The presence of an a,b-unsaturated alcohol moiety on the sesquiterpene side chain is also essential for the activity (Nkunya et al., 1990a).

Despite their novel structures, both the benzopyranyl sesquiterpenes, lucidene (13) and tanzanene (14) isolated from U. lucida ssp. Lucida (Weenen et al., 1990c) and U. tanzaniae, respectively (Weenen et al., 1991) and the schefflerins 15 and 16 from U. scheffleri (Nkunya et al., 1990b) are virtually inactive.

The three cyclohexene epoxides, (+) -pandoxide (17), (+)-b-senepoxide (18) and (-)-pipoxide (19), isolated from U. pandensis (compound 18 was also isolated from U. faulknerae), are weakly active. However, these compounds have been found to possess marked antibacterial, antifungal and antitumour activities (Nkunya et al., 1986).

We would like to emphasize that the compounds isolated in our investigations were the major ones. We are presently investigating whether more active minor components are present and whether these compounds can be isolated.

Conclusion

Our studies have indicated that most of the plants which are used for the treatment of malaria show at least some activity against the multidrug resistant K1 strain of P. falciparum malaria parasites. This, thus verifies the scientific basis for the traditional uses of these plants. However, these studies are only preliminary. More investigations for the in vivo activity and toxicity of the active plant extracts and pure compounds, are required for any definitive conclusions.

The results from our studies, and those reported by others, indicate that most of the active components are weakly, or medium polar compounds, which are soluble in petroleum ether or chloroform. However, in traditional medicines, water is the solvent which is used to prepare the extracts and concoctions. This is obviously so because the traditional healer has only water as the solvent for the preparation of his medicines. Thus in most cases the active ingredients in traditional medicines may be in minute concentrations, due to their low solubility in water. Therefore, larger quantities of these medicines are invariably needed for any curative effects. This appears to be the general practice with traditional medical practitioners.

The lack of suitable solvents means that many useful plants may not show any curative properties in traditional medicines, despite some of them containing highly potent compound(s), albeit in minute quantities. Therefore this calls for a massive scientific evaluation of plants so that should there be any potent, but minor component(s) in these plants, they should be characterised, so that efforts to synthesize them, or their analogues, can be initiated, with the objective of getting the compounds in larger quantities.

Acknowledgements

Financial support for this research, for which we are grateful, was obtained from the University of Dar es Salaam, the Norwegian Agency for International Development (NORAD), the Netherlands Universities Foundation for International Cooperation (NUFFIC), and the German Academic Exchange Service (DAAD). We are also grateful to the following people for providing spectral facilities: Prof. Dr. H. Achenbach (University of Erlangen, Germany); Prof. Dr. B. Zwanenburg (University of Nijmegen, The Netherlands); Prof. Dr. P. Waterman (University of Strathclyde, U.K.) and Dr. J. Wijnberg (University of Wageningen, The Netherlands). The plants used in this study were located and identified by Mr. L. B. Mwasumbi (The Herbarium, Botany Department, University of Dar es Salaam). We are grateful to Mr. F. Sung'hwa of the Department of Chemistry, University of Dar es Salaam who skillfully carried out most of the extractions and isolations of the pure compounds.

References

Bray, D.H., M.J. O'Neill, J.D. Phillipson, and D.C. Warhurst. (1987). J. Pharmac. Pharmacol., 39 (Suppl.): 85.

Breman, J.G. and C.C. Campbell. (1984). Bull. WHO. Geneva, 66: 611.

Chan, K.L., M.J. O'Neill, J.D. Phillipson, and D.C. Warhurst. (1986). Planta Med., 52: 105.

Farnsworth, N.R. (1984). In "Natural Products and Drug Development:, Krogsagaard - Larsen, P.; Brogger P.; Christensen, S. and Kofod, H. (Eds), Munksgaard, Copenhagen: 17.

Fandeur, T., C. Moretti, and J. Polonsky. (1985). Planta Med., 51: 20.

Gavagan, H. (1988). New Scientist, 28.

Hostettmann, K. (1990). Personal Communication.

Khan, M.R., G. Ndaalio, M.H.H. Nkunya, H. Wevers, and A.N. Sawhney. (1980). Planta Med. (Suppl.): 91.

Khan, M.R. and M.H.H. Nkunya. (1990). Proc. Internat. Conf. Trad. Med. Plants, Arusha (Tanzania), Feb. 19-23, 1990, these proceedings.

Kinabo, L.S. (1989). Chemical Studies of some Tanzanian antimalarial plants, M.Sc. Thesis, University of D'Salaam.

Kokwaro, J.O. (1976). Medicinal Plants of East Africa, East African Literature Bureau, Nairobi.

Leboeuf, M., A. Cave, P.K. Bhaumik, B. Mukherjee, and R. Mukherjee. (1982). Phytochemistry, 21: 2783.

Marandu, C.J.O. (1990). Isolation and identification of antimalarials and other constituents from Hoslundia opposita (Labiatae), M.Sc. Dissertation, University of Dar es Salaam.

Mgani, Q.A. (1990). Chemical studies of less polar constituents of Tanzania medicinal plants with antimalarial activity, M.Sc. Thesis, University of Dar es Salaam.

Nkunya, M.H.H. (1985). J. Nat. Prod., 48: 999.

Nkunya, M.H.H. and H. Weenen. (1986). Proc. 3rd. Internat. Chem. Conf. Africa, Lome (Togo). AFSAU and University of Benin (Togo): 313.

Nkunya, M.H.H., H. Weenen, and N.J. Koyi. (1987a). Phytochemistry, 26: 2402.

Nkunya, M.H.H., H. Weenen, N.J. Koyi, L. Thus, and B. Zwanenburg. (1987b). Phytochemistry, 26: 2563.

Nkunya, M.H.H. and H. Weenen. (1989). Phytochemistry, 28: 2217.

Nkunya, M.H.H., H. Weenen, D.H. Bray, Q.A. Mgani, and L.B. Mwasumbi, (1991). Planta Med. (in press).

Nkunya, M.H.H., H. Achenbach, C. Renner, R. Waibel, and H. Weenen. (1990). Phytochemistry 29: 1261.

O'Neill, M.J, D.H. Bray, P. Boardman, J.D. Philipson, D.C. Warhurst, W. Peters, and M. Suffness. (1986). Anitmicrob. Agents Chemother, 30: 101.

Phillipson, J.D. (1990). Personal Communication.

Pravanand, K., W. Nutaleum, T. Dechatiwongsen, K.L. Chan, M.J. O'Neill, J.D. Phillipson, and D.C. Warhurst. (1986). Planta Med., 52: 108.

Sawhney, A.N., M.R. Khan, G. Ndaalio, M.H.H. Nkunya, and H. Wevers. (1978a). Pakistan J. Sci. Ind. Res., 21: 193.

Sawhney, A.N., M.R. Khan, G. Ndaalio, M.H.H. Nkunya, and H. Wevers. (1978b). Pakistan J. Sci. Ind. Res., 21: 189.

Spencer, C.F., F.R Koninszy, E.F. Rogers, et al., (1947). Lloydia, 10: 145.

Sterling, L. (1977). Tanzanian Doctor, Heinemann Educational Books (East Africa) Ltd. Nairobi: 28.

Thaithong, S., G.H. Beale, and M. Chutmongkonkul. (1983). Trans. Roy, Soc. Trop. Med. Hyg., 77: 228.

Trager, W., and J. Polonksy. (1981). Am. J. Trop. Med. Hyg., 30: 531.

Underson, W.T. (1986). Africa Events (Science and Technology): 39.

Weenen, H., M.H.H. Nkunya, D.H. Bray, L.B. Mwasumbi, L.S. Kinabo, and V.A.E.B. Kilimali. (1990a): Planta Med., 56: 368.

Weenen, H., M.H.H. Nkunya, D.H. Bray, L.B. Mwasumbi, L.S. Kinabo, and V.A.E.B. Kilimali, and J. Wijnberg. (1990b): Planta Med., 56: 371.

Weenen, H., M.H.H. Nkunya, A. Abdul El-Fadl, S. Harkema, and B. Zwanenburg. (1990c). J. Org. Chem., 55: 5107.

Weenen, H., M.H.H. Nkunya, Q.A. Mgani, H. Anchenbach, M A. Posthumus, and R-Waibel. (1991). J. Org. Chem (in press)

WHO Report (1984): Development of Health Programmes Washington D.C.: 50.

WHO Report (1988): Disease Prevention and Control: 157.

WHO Research Activities, Biennium 1982/83: Malaria Chemotherapy: 113.

WHO Technical Report Series. (1964). Advances in Malaria Chemotherapy, 711: 142.

Xu-Ren Shen, Zhu Quiao-Zhen and Xie Yu Uan. (1985). J. Ethanopharmacol., 14: 233.

Table 1: Antimalarial activity of extracts of Tanzanian plants

Family	Species	Part useda	Activityb c
			PE	CH2Cl2	MeOH
Amarylidaceae	Crinum stuhlmannii	W.P.	N.D.	N.D.	**
	C. portifolium	W.P.	N.D.	N.D	-
	C. papilosum	W.P.	N.D.	N.D.	***
	Scadoxus multiflorus	W.P.	N.D.	N.D.	**
Anacardiaceae	Ozoroa insignis	R.B.	***	***	-
	Sclerocarya cafra	S.B.	-	-	-
	Sorindeia madagascariensis	R.B.	*	*	-
Annonaceae	Enantia kumeriae	R.B.	**	**	***
	Uvaria dependens Eng&Diels	R.B.	***	*	-
		S.B.	*	***	-
		leaves	*	*	-
	U. faulknerea Verdc.	R.B.	*	*	-
		S.B.	**	**	*
		leaves	-	*	-
	U. kirkii Hook. f.	R.B.	***	**	-
		S.B.	***	***	-
		leaves	-	*	-
	U. leptocladon Oliv.	R.B.	***	***	**
		S.B.	***	***	*
		leaves	-	*	*
	U. lucida ssp. lucida Benth.	R.B.	***	****	**
		S.B.	***	****	****
		leaves	**	***	*
	Uvaria sp. (Pande)	R.B.	****	***	*
		S.B.	****	***	*
		leaves	*	**	**
	U. pandensis Verdc.	R.B.	*	**	*
		S.B.	**	***	-
		leaves	*	*	*
	U. scheffleri Diels.	R.B.	**	****	****
		S.B.	**	**	*
		leaves	**	**	*
	U. tanzaniae Verdc.	R.B.	**	***	**
		S.B.	***	***	**
Apocynaceae	Rauvolfia mombasiana	R.B.	*	***	***
		S.B.	N.D.	N.D.	-
Araliaceae	Cussonia arborea	R.B.	***	***	-
Bignoniaceae	Kigelia africana	S.B.	-	***	-
		leaves	-	-	*
Caesalpinaceae	Caesalpinia bonduc	W.P.	N.D.	N.D.	-
	Cassia abbreviata	R.B.	**	*	*
	C. occidentalis	W.P.	-	-	-
	Tamarindus indica	fruits	N.D.	N.D.	-
Celastraceae	Catha edulis	aerial	N.D.	N.D.	-
Compositae	Artemisia afra	R.B.	**	***	*
		aerial	***	***	*
	Conyza pyrrhopappa	leaves	*	***	**
	Crassocephalum bojeri	aerial	*	***	**
	Tridax procumbens	W.P.	*	*	-
	Vernonia amygdalina	leaves	N.D.	N.D.	-
	V. colorata	R.B.	*	**	*
		S.B.	-	**	-
		leaves	-	**	-
Cyperaceae	Cyperus rotundus	tubers	***	****	***
		aerial	N.D.	*	N.D.
Ebenaceae	Diospyros natalensis	R.B.	**	*	N.D.
	D. zombensis	R.B.	*	*	N.D.
	D. greenwayii	R.B.	-	**	N.D.
		S.B.	-	**	N.D.
		leaves	-	**	N.D.
Euphorbiaceae	Bridelia cathartica	R.B.	*	**	-
	Clutia robusta	R.B.	-	-	-
	Margaritaria discoidea	R.B.	***	***	*
Guttiferae	Vismia orientale	S.B.	N.D.	N.D.	-
		leaves	N.D.	N.D.	-
Labiatae	Hoslundia opposita	R.B.	****	***	*
		S.B.	**	-	-
Lauraceae	Ocotea usambarensis	R.B.	***	***	*
Leguminosae	Acacia clavigera	S.B.	*	*	-
	Albizia anthelmintica	S.B.	-	-	-
	Piliostigma thonningii	S.B.	*	*	***
		leaves	*	*	***
Meliaceae	Azadirachta indica	S.B.	N.D.	N.D.	-
		leaves	*	**	-
	Entandrophragma bussei	S.B.	***	***	*
Myrtaceae	Psidium guajava	leaves	***	*	**
Olacaceae	Ximenia caffra	leaves	-	*	*
Plantaginaceae	Plantago major	W.P.	*	***	-
Rhizophoraceae	Anisophylia obtusifolia	R.B.	****	-	-
		S.B.	-	*	-
Rosaceae	Parinari exelsa sabin	S.B.	***	***	-
Rubiaceae	Crossopterix febrifuga	S.B.	*	*	*
	Gardenia jovis-tonantis	S.B.	N.D.	N.D.	-
		leaves	N.D.	N.D.	-
		fruit	-	**	-
	Vangueria infausta	R.B.	-	***	***
		S.B.	N.D.	N.D.	*
Rutaceae	Clausena anisata	R.B.	-	*	-
		leaves	*	**	*
	Todalia asiatica	R.B.	**	-	***
		S.B.	***	*	***
	Zanthoxylum gilletii	R.B.	***	***	**
		R.B.	**	**	***
	Z. xylubeum	S.B.	*	*	*
Tiliaceae	Grewia egglingii	S.B.	**	N.D.	N.D.
	G. forbesii	leaves	-	*	*
Verbenaceae	Lantana camara	R.B.	****	***	*
Zygophyaceae	Balanites aegyptica	S.B.	-	***	**